Polyanionic heteropolysaccharide biopolymers

ABSTRACT

Growth of Acinetobacter Sp. ATCC 31012 on various substrates and under varying conditions has been used to produce two classes of extracellular microbial protein-associated lipopolysaccharides (the &#34;emulsans&#34;) which, on a weight-for-weight basis, are probably the most efficient emulsifiers discovered and which possess certain characteristics that permit these unique extracellular microbial lipopolysaccharides to be widely used in cleaning oil-contaminated vessels, oil spill management, and enhanced oil recovery by chemical flooding. Emulsans and apoemulsans, both of which biopolymers are strongly anionic, exhibit a high degree of specificity in the emulsification of hydrocarbon substrates which contain both aliphatic and cyclic components. In addition, these extracellular microbial polysaccharides as well as their O-deacylated and N-deacylated derivatives are adsorbed on and capable of flocculating aluminosilicate ion-exchangers, such as kaolin and bentonite.

This is a division of application Ser. No. 012,971, filed Feb. 22, 1979,incorporated by reference, now abandoned.

TABLE OF CONTENTS

1. Introduction

2. Background of the Invention

3. Summary of the Invention

4. Nomenclature

5. Brief Description of the Drawings

6. Production of Emulsans and Apoemulsans

6.1 Acinetobacter Sp. ATCC 31012

6.2. Fermentation Media

6.2.1. Utilizable Carbon Sources

6.2.2. Additional Nutrients

6.2.3. Divalent Cations

6.3. Fermentation Process Conditions

6.3.1. Aeration

6.3.2. Agitation

6.3.3. Temperature and pH

6.3.4. Defoaming

6.4. Extracellular Production of Emulsans

6.4.1. Standard Assay for Emulsifying Activity

6.4.2. Extracellular Production of α-Emulsans

6.4.3. Extracellular Production of β-Emulsans

6.4.4. Distribution of Emulsifying Activity in Fractions of GrowthCulture

6.5. Deproteinization

6.6. Isolation and Purification

6.6.1. Heptane Partitioning

6.6.2. Ammonium Sulfate Precipitation

6.6.3. Quaternary Ammonium Salt Precipitation

7. Chemical and Physical Properties of Emulsans and Apoemulsans

7.1. Preparation of Samples for Analytical Characterization

7.1.1. Preparation of Emulsan

7.1.2. Preparation of Apoemulsan Samples

7.1.3. Ammonium Sulfate Fractionation of Apo-α-Emulsan

7.1.4. Quaternary Ammonium Salt Precipitation of Apo-α-Emulsan

7.2. Chemical Characterization

7.2.1. Chemical Composition of Emulsans and Apoemulsans

7.2.2. Alkaline Hydrolysis of Emulsans and Apoemulsans

7.2.3. Acid Hydrolysis of Apoemulsans and of Proemulsan

7.2.4. Identification of Sugar Components

7.3.5. Identification of Fatty Acids

7.3. Physical Characterization

7.3.1. Intrinsic and Reduced Viscosity

7.3.2. Sedimentation Velocity Analysis

7.3.3. Estimation of Molecular Weight

7.3.4. Spectral Properties

7.4. Conclusions on Structure

7.5. Variations in Structure

7.6. Immunological Characterization

8. Emulsifying Properties

8.1. Kinetics of Emulsan-Induced Emulsion Formation

8.2 Effect of pH and Salt Concentration on Emulsion Formation

8.3. Stability of Emulsan-Induced Emulsions

8.4. Lowering of Oil/Sea Water Interfacial Tensions

9. Specificity of the Hydrocarbon Substrate

9.1. Emulsification of Petroleum Fractions

9.2. Emulsification of Pure Hydrocarbons

9.3. Emulsification of Mixtures of Pure Hydrocarbons

9.4. Effect of Addition of Aliphatic and Aromatic Compounds onEmulsification of Petroleum Fractions

10. Summary of Differences Between α-Emulsans and β-Emulsans

10.1. Differences in Yield

10.2. Differences in Structure

10.3. Differences in Emulsifying Activity

11. Sorptive Properties of Emulsans and Their Derivatives on SolidSubstrates

11.1. Non-Adsorption on Sand and Limestone

11.2. Adsorption on Aluminosilicate Clays

11.3. Flocculation of Clays

11.4. Relationship of Flocculation to Breaking Oil/Water Emulsions

12. Environmental and Energy-Related Uses

13. Examples

13.1. Preparation of α-Emulsan from Ethanol in Fresh Water Media

13.2. Preparation of α-Emulsan from Ethanol in Sea Water Media

13.3. Preparation of α-Emulsan from Sodium Palmitate

13.4. Preparation of α-Emulsan from Dodecane

13.5. Preparation of β-Emulsan from Hexadecane

13.6. Preparation of Apo-α-Emulsan

13.7. Preparation of Apo-β-Emulsan

13.8. Preparation of ψ-Emulsan

13.9. Preparation of Proemulsan

13.10. Purification of α-Emulsan by Precipation with Ammonium Sulfate

13.11. Purification of α-Emulsan by Precipation with Quaternary AmmoniumSalts

13.12. Purification of β-Emulsan by Heptane Partitioning

13.13. Ammonium Sulfate Fractionation of Apo-α-Emulsan

13.14. Emulsification of Petroleum Fractions by α-Emulsans andβ-Emulsans

13.15. Emulsification of Mixtures of Petroleum Fractions and PureHydrocarbons by Emulsan

13.16. Cleaning Oil-Contaminated Vessels

13.17. Effect of Mobility Control Polysaccharides on Emulsion Formationwith Emulsan

13.18. Adsorption of Emulsans on Clays

13.19. Flocculation of Clays by Emulsans

13.20. Flocculation of Clays by Proemulsans

13.21. Breaking Emulsan-Induced Emulsions

13.22. Removal of Oil from Sand by Emulsan

13.23. Removal of Oil from Limestone by Emulsan

1. INTRODUCTION

This invention relates to extracellular microbial polysaccharides(herein generically called "emulsans") produced by Acinetobacter Sp.ATCC 31012 and, more particularly, to a new class of extracellularmicrobial protein-associated lipopolysaccharides (herein collectivelycalled "α-emulsans") produced by this organism and its mutants orrecombinants. The invention further relates to the deproteinizedlipopolysaccharides (herein collectively called "apoemulsans") obtainedfrom such emulsans, as well as to the divalent metal, ammonium andquaternary ammonium salts of such emulsans and apoemulsans. Theseextracellular microbial polysaccharides, which include both the emulsansand apoemulsans and their respective salts, are among the most efficientoil-in-water emulsifiers ever discovered and possess a high degree ofspecificity in both fresh water and sea water for emulsifying thosehydrocarbon substrates which contain both aliphatic and aromatic orcyclic components, properties which make these unique bioemulsifiersideal for use in cleaning oil-contaminated vessels, oil spillmanagement, and enhanced oil recovery by chemical flooding.

2. BACKGROUND OF THE INVENTION

A wide variety of petroleum-degrading microorganisms has been found tobring about the formation of oil-in-water emulsions while growing onhydrocarbons. These emulsions are microbiological in origin and appearto be mediated either by the cells themselves or by the production ofextracellular emulsifying agents. For example, the growth ofMycobacterium rhodochrous NCIB 9905 on n-decane yields an emulsifyingfactor which was reported by R. S Holdom et al. [J. Appl. Bateriol., 32,448 (1969)] to be a nonionic detergent. J. Iguchi et al. [Agric Biol.Chem., 33, 1657 (1969)] found that Candida petrophilium produced anemulsifying agent composed of peptides and fatty acid moieties, while T.Suzuki et al. [Agric. Biol. Chem., 33, 1619 (1969)] found trehaloselipid in the oil phase of culture broths of various strains ofArthrobacter, Brevibacterium, Corynebacterium and Norcardia.

Torulopsis gropengiesseri was found to produce a sophorose lipid, whilerhamnolipids are reported by K. Hisatsuka et al. [Agric. Biol. Chem.,35, 686 (1971)] to have been produced by Pseudomonas aeruginosa strainS7B1 and by S. Itoh et al. [Agric. Biol. Chem., 36, 2233 (1971)] to havebeen produced by another P. aeruginosa strain, KY4025. The growth ofCorynebacterium hydrocarbolastus on kerosene was reported by J. E. Zajicand his associates [Dev. Ind. Microbiol., 12, 87 (1971); Biotechnol.Bioeng., 14, 331 (1972); Chemosphere, 1, 51 (1972); Crit. Rev.Microbiol., 5, 39 (1976); U.S. Pat. No. 3,997,398] to produce anextracellular heteropolysaccharide which, among other properties,emulsified kerosene, Bunker C fuel oil and other fuel oils.

In U.S. Pat. No. 3,941,692, we described the use of an Arthrobacterspecies, RAG-1 (which, upon deposit with the American Type CultureCollection, has been designated as Arthrobacter Sp. ATCC 31012 and isnow known to have been an Acinetobacter species and has beenredesignated as Acinetobacter Sp. ATCC 31012) to clean oil-contaminatedtank compartments by allowing the organism to aerobically grow on theoily wastes in such tanks using sea water containing added nutrients.During that microbially-induced cleaning process, the organism appearedto secrete one or more dispersants during the fermentation, since thecell-free fermentation medium was also effective in cleaning waste oilfrom such tanks.

Further studies on the microbial degradation of crude oil by thisorganism [Appl Microbiol., 24, 363 (1972); Appl. Microbiol., 30, 10(1975)], showed that RAG-1 emulsified the oil during exponential growth,probably by producing an extracellular emulsifying agent which acted tobreak up the oil droplets into smaller units and thereby produce newsurface area, necessary for the increasing cell population. At the 1stInternational Congress for Bacteriology held Sept. 2-7, 1973 [Int.Assoc. Microbiol. Soc. Abstracts, Vol. II, p. 201], we reported thatthis extracellular emulsifying agent had been partially purified fromstationary phase cultures of RAG-1 growing on 0.4% hexadecane, 0.075 Murea and 5.8 mM dibasic potassium phosphate in sea water. The partiallypurified extracellular emulsifying agent was obtained by extensivelydialyzing and then lyophilizing the cell-free fermentation broth,yielding 0.25 mg per ml of culture fluid of a dry powder which wascapable of forming a stable oil-in-water emulsion with 40 times itsweight of crude oil.

Notwithstanding the many publications on the subject, however,microbially-induced emulsification of oil is poorly understood from bothmechanistic and teleological points of view. Microorganisms can utilizecrude oil as a substrate for growth with or without concomitant oilemulsification. Where emulsification has occurred because of theproduction of extracellular emulsifying agents, in general thepreparations have not been purified sufficiently to identify the activecomponents. In sum, none of these extracellular bioemulsifiers has beenwell characterized and very little is known about their chemicalproperties, mode of action or biological function.

3. SUMMARY OF THE INVENTION

The present invention is based upon part of a multitude of discoveriesmade in connection with further work done on the bioemulsifiers producedby Acinetobacter Sp. ATCC 31012, among the most important of whichdiscoveries were:

Firstly, that the Acinetobacter bioemulsifier previously produced bygrowing Acinetobacter Sp. ATCC 31012 (also known as strain RAG-1) oncrude oil or hexadecane is an extracellular microbial protein-associatedlipopolysaccharide (which we have herein called "β-emulsan" and giventhe common name "protoemulsans"), in which the lipopolysaccharide is anN- and 0-lipoacylated heteropolysaccharide made up of major amounts ofD-galactosamine and an aminouronic acid, the 0-lipoacyl portion of thelipoheteropolysaccharide containing from 2 to 3 percent by weight ofvarious fatty acid esters in which (a) the fatty acids contain fromabout 10 to about 18 carbon atoms; and (b) less than 50 percent byweight of such fatty acids are composed of 2-hydroxydodecanoic acid and3-hydroxydodecanoic acid;

Secondly, that growth of Acinetobacter Sp. ATCC 31012 on certain otherhydrocarbons or on certain oxygen-containing carbonaceous compounds asthe primary assimilable carbon source yields a significantly differentextracellular microbial protein-associated lipopolysaccharide (which wehave herein called "α-emulsans" and given the common name"neoemulsans"), in which the lipopolysaccharide is also an N- and0-lipoacylated heteropolysaccharide made up of major amounts ofD-galactosamine and an aminouronic acid, but in which the 0-lipoacylportion of the lipoheteropolysaccharide contains at least 5 percent byweight (and, more often, between 7 to 14 percent by weight andoccasionally as high as 19 percent by weight) of various fatty acidesters in which (a) the fatty acids contain from about 10 to about 18carbon atoms which are usually distributed in different ratios thanthose in the lower-ester β-emulsans; and (b) about 50 percent by weightor more of such fatty acids are composed of 2-hydroxydodecanoic acid and3-hydroxydodecanoic acid;

Thirdly, that α-emulsans are much more effective than β-emulsans in theemulsification of various crude oils and gas-oils and, in some instances(such as the emulsification of Bunker C fuel oil), efficiently formstable emulsions where β-emulsans have no effect;

Fourthly, that both α-emulsans and β-emulsans exhibit specificity in theemulsification of various types of hydrocarbons;

Fifthly, that upon deproteinization of the emulsans all of theemulsifying activity is in the respective N- and 0-lipoacylheteropolysaccharides (which we have herein generically called"apoemulsans", and specifically called "apo-α-emulsan" or"apo-β-emulsan" depending upon the particular emulsan from which suchdeproteinized derivative was formed);

Sixthly, that base hydrolyses of α-emulsan and β-emulsan under mildconditions yield a common derivative (which we have herein called"ψ-emulsans" and given the common name "pseudoemulsans") which retainsabout 50 percent of the emulsifying activity of the α-emulsans, thestructure of which ψ-emulsans is the N-acylatedpoly[D-galactosamine/aminouronic acid] in which (a) the amount of fattyacid esters is between 0 and 1 percent by weight of the polysaccharide;and (b) part of the N-acyl groups are 3-hydroxydodecanoyl groups;

Seventhly, that base hydrolyses of α-emulsan and β-emulsan under strongconditions yield a derivative (which we have herein called"proemulsans") which has no emulsifying activity and which isstructurally a partially N-acylated poly[D-galactosamine/aminouronicacid];

Eighthly, that antibodies prepared against β-emulsan cross-react in anidentical fashion with α-emulsan, apo-α-emulsan, apo-β-emulsan,ψ-emulsan and proemulsan, indicating that the emulsans and theirdeproteinized and partially deacylated derivatives have approximatelythe same polymer backbones, which are poly[D-galactosamine/aminouronicacid] polymers;

Ninthly, that the emulsans and their respective deproteinizedderivatives are not affected by high concentrations of sodium chloridebut require small amounts (from 1 to 100 mM and preferably from 5 to 40mM) of at least one divalent cation, such as magnesium, calcium ormanganese, to function effectively as emulsifying agents for hydrocarbonsubstrates, which divalent cations are present in sea water, connatewater and most "hard" water but must be added to "soft" water;

Tenthly, that emulsans on a weight-for-weight basis are probably themost efficient oil-in-water emulsifiers discovered and, moreover,possess certain characteristics that permit these unique extracellularmicrobial polysaccharides to be widely used in cleaning oil-contaminatedvessels, oil spill management, and enhanced oil recovery by chemicalflooding;

Finally, that the emulsans and their deproteinized and deacylatedderivatives are strongly adsorbed onto aluminosilicate ion-exchangersand are unusually efficient bioflocculents which may be used to mediateflocculation of various types of aluminosilicate clays, such as kaolinand bentonite.

Based on some of these discoveries, the invention provides several newclasses of extracellular microbial lipopolysaccharides and theirderivatives selected from the group consisting of

(a) the extracellular microbial protein-associated lipopolysaccharides(herein collectively called "α-emulsans") produced by Acinetobacter Sp.ATCC 31012 and its mutants, in which the lipopolysaccharide components(herein collectively called "apo-α-emulsans") are completely N-acylatedand partially O-acylated heteropolysaccharides made up of major amountsof D-galactosamine and an aminouronic acid, such apo-α-emulsanscontaining at least 5 percent by weight of fatty acid esters in which(1) the fatty acids contain from about 10 to about 18 carbon atoms; and(2) about 50 percent by weight or more of such fatty acids are composedof 2-hydroxydodecanoic acid and 3-hydroxydodecanoic acid;

(b) the deproteinized extracellular microbial lipopolysaccharides(herein collectively called "apo-α-emulsans") obtained from theα-emulsans produced by Acinetobacter Sp. ATCC 31012 and its mutants, theapo-α-emulsans being completely N-acylated and partially O-acylatedheteropolysaccharides made up of major amounts of D-galactosamine and anaminouronic acid, the apo-α-emulsans containing at least 5 percent byweight of fatty acid esters in which (1) the fatty acids contain fromabout 10 to about 18 carbon atoms; and (2) about 50 percent by weight ormore of such fatty acids are composed of 2-hydroxydodecanoic acid and3-hydroxydodecanoic acid;

(c) the deproteinized extracellular microbial polysaccharides (hereincollectively called "apo-β-emulsans") obtained from the β-emulsansproduced by Acinetobacter Sp. ATCC 31012 and its mutants, theapo-β-emulsans being completely N-acylated and partially O-acylatedheteropolysaccharides made up of major amounts of D-galactosamine and anaminouronic acid, the apo-β-emulsans containing not more than 5 percentby weight of fatty acid esters in which (i) the fatty acids contain fromabout 10 to about 18 carbon atoms; and (2) less than 50 percent byweight of such fatty acids are composed of 2-hydroxydodecanoic acid and3-hydroxydodecanoic acid;

(d) the O-deacylated extracellular protein-associated microbialpolysaccharides (herein collectively called the "ψ-emulsans") obtainedfrom the emulsans produced by Acinetobacter Sp. ATCC 31012 and itsmutants, the protein-free components of such ψ-emulsans being completelyN-acylated heteropolysaccharides made up of major amounts ofD-galactosamine and an aminouronic acid and containing from 0 to 1percent by weight of fatty acid esters in which, when present, the fattyacids contain from about 10 to about 18 carbon atoms;

(e) the deproteinized O-deacylated extracellular microbialpolysaccharides (herein collectively called the "apo-ψ-emulsans")derived from either α-emulsans, β-emulsans, ψ-emulsans, apo-α-emulsansor apo-β-emulsans, the apo-ψ-emulsans being completely N-acylatedheteropolysaccharides made up of major amounts of D-galactosamine and anaminouronic acid and containing from 0 to 1 percent by weight of fattyacid esters in which, when present, the fatty acids contain from about10 to about 18 carbon atoms;

(f) the deproteinized O-deacylated extracellular microbialpolysaccharides (herein collectively called the "proemulsans") derivedfrom either α-emulsans, β-emulsans, ψ-emulsans, apo-α-emulsans,apo-β-emulsans or apo-ψ-emulsans, the proemulsans beingpoly[D-galactosamine/amino uronic acid] biopolymers in which (1) none ofthe hydroxy group are acylated; and (2) from none to all of the aminogroups are acylated; and

(g) the divalent metal, ammonium and quaternary ammonium salts of suchα-emulsans, apo-α-emulsans, apo-β-emulsans, ψ-emulsans, apo-ψ-emulsansand proemulsans.

The invention further provides emulsifying agents comprising an aqueoussolution in sea water or fresh water containing from about 10 mcg/ml toabout 20 mg/ml of such α-emulsans, and from about 1 to about 100 mM ofat least one divalent cation. Using the data contained herein, theemulsifying agents of the invention may be used, among other things, (1)for cleaning hydrocarbonaceous residues, including residual petroleum,from tankers, barges, storage tanks, tank cars and trucks, pipelines andother containers; (2) for cleaning oil spills which are floating on thesea or which have been washed ashore or which are deposited on land; and(3) for the enhanced recovery of oil by chemical flooding techniques,particularly with respect to those petroleum reservoirs located in sandor sandstone or limestone formations.

The invention also contemplates those polyanionic heteropolysaccharidesbiopolymers which are produced microbiologically (regardless of theorganism used) or by semi-synthetic techniques (such as by anzymaticactivity) and in which (a) substantially all of the sugar moieties areN-acylated amino sugars, a portion of which is N-acylatedD-galactosamine and another portion of which is an aminouronic acid(such as D-galactosamineuronic acid, D-glucoseamineuronic acid), a partof the N-acyl groups of such heteropolysaccharide beingN-(3-hydroxydodecanoid) groups; and (b) at least 0.2, and preferablyfrom about 0.5 to about 0.75 micromoles per milligram of suchheteropolysaccharide consists of fatty acid esters in which (1) thefatty acids contain from about 10 to about 18 carbon atoms, and (2)about 50 percent by weight of such fatty acids are composed of2-hydroxydodecanoic acid and 3-hydroxydodecanoic acid.

4. NOMENCLATURE

A new lexicon has been used herein to identify and refer to the varioustypes of extracellular microbial polysaccharides and theirsemi-synthetic derivatives which are derived from Acinetobacter Sp. ATCC31012 and its mutants. These new words are "emulsans", "α-emulsans","β-emulsans", "ψ-emulsans", "apoemulsans", "apo-α-emulsans","apo-β-emulsans", "apo-ψ-emulsans", and "proemulsans", which are definedas follows:

The name "emulsans", which reflects the polysaccharide structure ofthese compounds and the exceptional emulsifying activity of thebiologically produced materials, has been created to identifygenerically those extracellular microbial protein-associatedlipoheteropolysaccharides produced by Acinetobacter Sp. ATCC 31012 andits mutants, which may be subdivided into the α-emulsans and theβ-emulsans. The name "apoemulsan", the prefix of which is derived fromthe Greek word απο meaning "from", has been created to identifygenerically those deproteinized lipopolysaccharides obtained from theemulsans.

The name "α-emulsans" defines those extracellular microbialprotein-associated lipopolysaccharides produced by Acinetobacter Sp.ATCC 31012 and its mutants in which the lipopolysaccharide components(i.e., without the associated protein) are completely N-acylated andpartially O-acylated heteropolysaccharides made up of major amounts ofD-galactosamine and an aminouronic acid, the lipopolysaccharidecomponents containing at least 5 percent by weight of fatty acid estersin which (1) the fatty acids contain from about 10 to about 18 carbonatoms; and (2) about 50 percent by weight or more of such fatty acidsare composed of 2-hydroxydodecanoic acid and 3-hydroxydodecanoic acid.It follows, therefore, that the deproteinized α-emulsan ar named"apo-α-emulsans".

The name "β-emulsans" defines those extracellular microbialprotein-associated lipopolysaccharides produced by Acinetobacter Sp.ATCC 31012 and its mutants in which the lipopolysaccharide components(i.e., without the associated protein) are completely N-acylated andpartially O-acylated heteropolysaccharides made up of major amounts ofD-galactosamine and an aminouronic acid, the lipopolysaccharidecomponents containing less than 5 percent by weight of fatty acid estersin which (1) the fatty acids contain from about 10 to about 18 carbonatoms; and (2) less than 50 percent by weight of such fatty acids arecomposed of 2-hydroxydodecanoic acid. The deproteinized β-emulsans arenamed "apo-β-emulsans".

The name "ψ-emulsans" defines the O-deacylated extracellularprotein-associated microbial polysaccharides obtained from the emulsans,the protein-free components of such ψ-emulsans being completelyN-acylated heteropolysaccharides made up of major amounts ofD-galactosamine and an aminouronic acid and containing from 0 to 1percent of fatty acid esters in which, when present, the fatty acidscontain from about 10 to about 18 carbon atoms. These protein-freecomponents are named "apo-ψ-emulsans", regardless of how they areprepared.

The name "proemulsans" defines the deproteinized O-deacylatedextracellular microbial polysaccharide in which thepoly[D-galactosamine/aminouronic acid] biopolymers are characterized by(1) none of the hydroxy groups being acylated; and (2) from none to allof the amino groups being acylated. The proemulsans have no emulsifyingactivity under the standard assay techniques described below.

From the data described herein, it is now known that the bioemusifierswhich were inherently formed in the experimental work previouslypublished concerning the growth of RAG-1 on crude oil or hexadecane wereβ-emulsans in which the lipopolysaccharide contained from 2 to 3 percentby weight of fatty acid esters. The β-emulsans, therefore have beengiven the common name "protoemulsans", the prefix of which is derivedfrom the Greek word προτο meaning "first".

The α-emulsans have been given the common name "neoemulsans", the prefixhaving derived from the Greek word ηεοζ meaning "new". Because theψ-emulsans have only about one-half the emulsifying activity of theα-emulsans, the ψ-emulsans have been given the common name"pseudoemulsans".

As used herein, the term "Acinetobacter Sp. ATCC 31012 and its mutants"refers not only to the organism (i.e., strain RAG-1) described below inSection 6.1 and its spontaneous and chemically- and physically-inducedmutants and recombinants which produce emulsans, but to allmicroorganisms (whatever the genus) derived by using recombinant DNAtechniques to insert genetic information from strain RAG-1 and suchmutants which are responsible for the production of the bioemulsifiersinto the DNA-based genetic code of such "recombined" microorganisms suchthat they are capable of biosynthesizing α-emulsans or β-emulsans (orthe apoemulsans), depending upon the primary assimilable carbon sourceused to grow the organism.

5. BRIEF DESCRIPTION OF THE DRAWINGS

To more fully comprehend the invention, reference should be made to theaccompanying drawings, in which

FIG. 1 is a graphical representation of the standard emulsifier assaydescribed below in Section 6.4.1, showing the relationship between theamount of emulsification which is obtained with gas-oil and with a 1:1(v/v) mixture of hexadecane/2-methylnaphthalene as a function of theemulsan concentration;

FIG. 2 is a graphical representation of the extracellular production ofα-emulsan during growth of Acinetobacter Sp. ATCC 31012 on an ethanolmedium, showing the relationship of the growth of the organism in suchmedium, the production of the bioemulsifier during such growth, and thechange of pH during such growth, all as a function of time;

FIG. 3 is a graphical representation of the extracellular production ofβ-emulsan during growth of Acinetobacter Sp. ATCC 31012 on a hexadecanemedium, showing the relationship of the growth of the organism in suchmedium and the production of the bioemulsifier during such growth, bothas a function of time;

FIG. 4 is a graphical representation of the changes which occur on saidhydrolysis of apo-α-emulsan, showing the relationship between the weightpercent of reducing power of the acid-hydrolyzed deproteinized0-lipoacylated heteropolysaccharide as a function of the duration ofhydrolysis;

FIG. 5 is a graphical representation of the relationship of the reducedviscosity of apo-α-emulsan as a function of ionic strength;

FIG. 6, which is subdivided into FIGS. 6A and 6B, is a graphicalrepresentation of the kinetics of the emulsan-induced emulsification ofgas-oil, showing the relationship between the emulsification of varyingconcentrations of gas-oil as a function of time for a givenconcentration of the bioemulsifier;

FIG. 7 is a graphical representation of the relationship between theamount of emulsification which is obtained 60 minutes after mixing inthe emulsan-induced emulsification of gas-oil as a function of gas-oilconcentration for a given concentration of the bioemulsifier;

FIG. 8 is a graphical representation of the relationship between theamount of emulsification which is obtained in the emulsan-inducedemulsification of gas-oil as a function of pH in fresh water and seawater in the presence and absence of magnesium ions;

FIG. 9 is a graphical representation of the relationship between theamount of emulsification which is obtained in the emulsan-inducedemulsification of gas-oil as a function of salt concentration;

FIG. 10, which is subdivided into FIGS. 10A and 10B, is a graphicalrepresentation of the relative stabilities of emulsan-induced emulsionsof gas-oil, showing the relationship between percentage change inemulsification as a function of standing time of the emulsion for agiven concentration of bioemulsifier and varying weight ratios ofgas-oil/bioemulsifier;

FIG. 11 is a graphical representation of the rate at which emulsifiedoil droplets rise as a function of the weight ratio of gas-oil tobioemulsifier for given concentrations of the bioemulsifier;

FIG. 12 is a graphical representation showing the relationship betweenthe interfacial tension of n-alkanes in sea water containing a givenconcentration of emulsan as a function of n-alkane chain length;

FIG. 13 is a graphical representation showing the relationship of theamount of emulsification which is obtained in the emulsan-inducedemulsification of various straight and branch chain alkanes as afunction of carbon number of such alkanes;

FIG. 14 is a graphical representation showing the relationship of theamount of emulsification which is obtained in the emulsan-inducedemulsification of various alkylcyclohexanes as a function of carbonnumber of such alkylcyclohexanes;

FIG. 15 is a graphical representation showing the relationship of theamount of emulsification which is obtained in the emulsan-inducedemulsification of various alkyl-substituted benzenes as a function ofcarbon number of such alkylbenzenes;

FIG. 16 is a graphical representation showing the relationship of theamount of emulsification which is obtained in the emulsan-inducedemulsification of mixtures of hexadecane and a particularmethylnaphthalene as a function of the volume percent of hexadecane insuch mixtures;

FIG. 17 is a graphical representation of the kinetics of adsorption ofemulsan on bentonite, showing the relationship between the amount ofemulsan remaining in solution as a function of time after a givenconcentration of emulsan is shaken with a given amount of bentonite;

and

FIG. 18 is a graphical representation of the kinetics of bentoniteflocculation by emulsan, showing the relationship between the amount ofclear upper layer which appears during sedimentation as a function oftime when a given amount of bentonite is dispersed in a standardizedcontrol solution containing no added bioemulsifier and in the samesolution containing a given concentration of emulsan and bothdispersions are allowed to settle.

6. PRODUCTION OF EMULSANS AND APOEMULSANS

Emulsans may be produced by aerobically growing Acinetobacter Sp. ATCC31012 or its mutants on an aqueous fermentation medium which contains(a) a growth-sustaining amount of a utilizable carbon source on whichthe organism will not only grow but will also produce the desiredemulsan (such as α-emulsan) rather than the low-ester protoemulsan; (b)growth-sustaining amounts of nitrogen- and phosphorous-containingcompounds to furnish these essential nutrients to the organism; and (c)from about 1 to about 100 mM of a divalent cation, such as magnesium,calcium or manganese, which must be added to the fermentation medium ifnot present. Apoemulsans, in turn, are produced by deproteinization ofthe emulsans in such manner that the lipoheterpolysaccharide does notundergo degradation.

The fermentation process may be conducted with automatic or manualcontrol in batch or continuous fermenters, using either fresh water orsea water media. Selection of suitable fermentation equipment may bemade from designs engineered to give the most efficient oxygen transferto the biomass at lowest operating cost. In addition to the stirred tankfermenters, other types of fermenters may be used, such as thin channelfermenters, tubular loop fermenters, film fermenters, recirculatingtower fermenters, deep shaft fermenters, and jet fermenters, the mostimportant criteria being efficiency in the fermentation process,especially with respect to oxygen transfer and power consumption.

Some of the more important process parameters for the production andpurification of α-emulsans and apo-α-emulsans are discussed in moredetail below.

6.1. ACINETOBACTER SP. ATCC 31012

The microorganism used to produce both neoemulsans and protoemulsansfrom utilizable carbon sources is Acinetobacter Sp. ATCC 31012 (alsoknown as strain RAG-1), which has been deposited previously with theAmerican Type Culture Collection, Rockville, Md. This organism, whichhas been described by A. Reisfeld et al., Appl. Microbiol., 24, 363(1972) as well as by U.S. Pat. No. 3,941,692, has the followingcharacteristics:

During the exponential growth phase the cells appear mostly as irregularshort rods, 0.9 to 1.2 by 1.5 to 3.0 mcm (mcm=10⁻⁶ m). The cells occuroften as V-shaped pairs, indicating snapping division. Occasionally, therods are slightly bent or swollen. Coccoid cells, approximately 1.2 mcmin diameter, are characteristic of stationary phase cultures. The cocciare gram-positive; the rods are gram-negative.

Agar colonies: circular, glistening and smooth, up to 5.0 mm indiameter; gelatin is liquified; starch is not hydrolyzed; indole andhydrogen peroxide are not produced; nitrites are produced from nitrateonly when the cells are grown in citrate medium containing potassiumnitrate; urease is not produced; catalase-positive; aerobic; hemolysisof rabbit blood agar; citrate can serve as the sole carbon and energysource; no acid from glucose, cellulose, maltose, lactose, rhamnose,sucrose or mannitol; optimum temperature 30° to 35° C.

The amount of inoculum used to initiate the fermentation will bedependent upon the type of fermentation equipment used. For optimumresults in batch-type stirred fermenters, growth should be initiatedwith late exponential cultures grown under similar fermentationconditions, preferably in an amount from about 1% to about 5% (v/v) ofthe fermentation medium.

6.2. FERMENTATION MEDIA 6.2.1. UTILIZABLE CARBON SOURCES

Even though it has previously been reported by A. Horowitz et al., Appl.Microbiol, 30, 10 (1975), that strain RAG-1 will grow on many differentcarbon compounds on sea water agar media supplemented with the carbonsource, such growth has no relationship with whether or not the organismwill produce any type of Acinetobacter bioemulsifier (which, whenproduced, usually occurs during the exponential growth phase), much lessthe high-ester α-emulsans. Moreover, even in those instances where theorganism does produce extracellular lipopolysaccharides, there does notappear to be any correlation between the structure of the utilizablecarbon source and what type of extracellular lipopolysaccharide will bebiosynthesized from such carbon source, whether the high-esterα-emulsans or the low-ester β-emulsans. For example, growth ofAcinetobacter Sp. ATCC 31012 on ethanol, sodium palmitate or dodecaneresults in the formation of α-emulsans with each such carbon source,with ethanol media yielding α-emulsans with the hightest ester contentin the lipoacyl portion of the lipoheteropolysaccharide, while growth ofthe organism under substantially identical conditions using pentadecane,hexadecane or heptadecane results only in the formation of β-emulsans.In general, where a utilizable carbon source can be transformed intoα-emulsans by the organism, the total yield of the extracellularlipopolysaccharide per liter of culture medium will be greater than whenthe organism produces β-emulsans from a different carbon source.

α-Emulsans produced by aerobically growing Acinetobacter Sp. ATCC 31012on ethanol media in accordance with the invention are unusuallyefficient bioemulsifiers, exhibiting a high degree of specificity inemulsifying those hydrocarbon substrates (such as crude oils, gas-oilsand Bunker C fuel oils) that contain both aliphatic and aromaticcomponents. For optimum results in batch-type stirred fermenters, theinitial media should contain about 1.25% to about 3% (v/v) andpreferably about 1.75% to about 2.5% (v/v) of ethanol, with make-upethanol added during the fermentation at a rate sufficient to sustainmaximum growth and α-emulsan formation, since the production ofα-emulsans by the organism has been found to occur during the growthperiod.

6.2.2. ADDITIONAL NUTRIENTS

Maximum growth of Acinetobacter Sp. ATCC 31012 on a utilizable carbonsource to produce α-emulsans or β-emulsans requires more thangrowth-sustaining amounts of one or more nitrogen-containing compoundsto furnish this essential nutrient to the organism and to enable theorganism to grow and to produce the biopolymer, which contains majoramount of two amino sugars. Additionally, phosphorus-containingcompounds are also essential nutrients. Suitable sources of availablenitrogen include ammonium salts, such as ammonium sulfate or ammoniumchloride; nitrates, such as ammonium nitrate or sodium nitrate; ororganic sources of available nitrogen, such as urea or soybean meal.Suitable sources of available phosphorous include dibasic potassiumphosphorus, monobasic potassium phosphate and the like. In addition,liquid fertilizers, such as 12-6-6 or 8-8-8, may serve as a source ofnitrogen and phosphorous nutrients for the growth of Acinetobacter Sp.ATCC 31012.

6.2.3. DIVALENT CATIONS

As shown below in the data set forth in Section 8.4, the emulsifyingactivity of both types of Acinetobacter bioemulsifiers is dependentabove pH 6 upon divalent cations, such as magnesium ions, calcium ionsor manganese ions. These divalent cations are present in sea water or"hard" water when fermentation media are prepared from such sources.When "soft" fresh water or distilled water are used to prepare thefermentation media, then small amounts of one or more salts of adivalent cation should be added to the fermentation media, theconcentration being such that the resultant culture media will containfrom about 1 to about 100 mM (and preferably from about 5 to about 40mM) of at least one divalent cation.

6.3. FERMENTATION PROCESS CONDITIONS

Maximum growth of Acinetobacter Sp. ATCC 31012 upon utilizable carbonsources to produce α-emulsans requires selection of the best conditionsof aeration, agitation, temperature and pH under which the highestpossible oxygen transfer can be obtained consistent with the physiologyof the organism. Discussed below are the best conditions which have beenfound for consistently producing 4 to 5 mg/ml of α-emulsans from ethanolmedia in conventional 60-liter stirred fermenters. These conditionsprobably will undergo subtle or pronounced changes to obtain higheryields upon large-scale production in fermenters specifically designedor adapted to give more efficient oxygen transfer at the lowest powerconsumption. Subsequent work on optimizing the process will, of course,focus on (a) consumption of the substrate, which is a function of thephysiology of Acinetobacter Sp. ATCC 31012 and its mutants; (b)consumption of oxygen, which is a function of oxygen diffusion to thecells which, in turn, will be influenced (i) by making the surfacethrough which the diffusion occurs as large as possible (i.e.,dispersing the gas phase as finely as possible in the liquid phase tocreate a large gas hold-up), (ii) by increasing the driving force of thediffusion (such as by increasing the pressure in the fermenter or byusing oxygen-enriched air), and (iii) by allowing the diffusion constantto be as high as possible (i.e., by minimally decreasing the diffusionconstant by the use of chemical antifoam agents); and (c) exothermicheat production, which necessitates a properly designed cooling systemon scale-up.

6.3.1. AERATION

Using 60-liter stirred fermenters with the fermentation medium andprocess conditions described below in Section 13.1, maximum productionof α-emulsans occurs when 15 liters of air per minute are passed throughthe 40 liters of fermentation medium, which corresponds to an oxygenflow rate of 189.6 millimoles per liter per hour. This oxygen flow rateis not limiting but can, if necessary, be increased to as high as 700millimoles per liter per hour, or even higher, with the more efficientlydesigned fermenters.

6.3.2. AGITATION

To promote maximum oxygen diffusion to the cell mass, the fermentationmedia must be agitated either by stirring or circulating the mediathrough the fermenter, depending upon the type of fermentation equipmentemployed. Using 60-liter stirred fermenters with the fermentation mediumand other process conditions described below in Section 13.1, maximumproduction of α-emulsans occurs when the medium is agitated at a rate of250 rpm. This value is not limiting but will be varied in the moreefficiently designed fermenters to achieve maximum oxygen transfer atthe lowest power consumption.

6.3.3. TEMPERATURE AND pH

Although the fermentation process may be conducted over a wide range oftemperatures, best results have been obtained in the production ofα-emulsans when the fermentation is conducted at 30° C. The pH of thefermentation medium should be maintained between 6 and 7, and preferablybetween 6.2 and 6.7 during the exponential growth phase, whichnecessitates the addition of sufficient base (preferably ammonia).

6.3.4. DEFOAMING

Stirred-tank fermentations of Acinetobacter Sp. ATCC 31012 on utilizablecarbon sources to produce α-emulsans invariably are accompanied byfoaming problems, which diminish the realizable yield of theextracellular lipopolysaccharide. Although many types of chemicaldefoamers may be used in the fermentation media, great care must betaken when adding chemical defoaming agents to keep the diffusionconstant as high as possible. Using the 60-liter stirred fermenters withthe fermentation medium and other process conditions described below inSection 13.1, maximum production of α-emulsans occurs when there wereautomatic pulse additions (whenever foam levels reached a predeterminedheight) of a silicone defoamant, preferably Dow-Corning 525(sterilizable), diluted 1:8. Upon scale-up of the fermentation process,it is expected that a combination of chemical and mechanical methodswill give optimum results in defoaming the nutrient solutions on whichα-emulsans will be produced from Acinetobacter Sp. ATCC 31012 and itsmutants.

6.4. EXTRACELLULAR PRODUCTION OF BIOEMULSIFIERS

Data is presented below with respect to both types of extracellularlipopolysaccharides (α-emulsans and β-emulsans) produced byAcinetobacter Sp. ATCC 31012 so that the similarities as well asdifferences between these biopolymers may be understood. Unless theparticular type of extracellular lipopolysaccharide produced by theorganism is identified by name, the phrase "Acinetobacter bioemulsifier"refers collectively to both α-emulsans and β-emulsans.

6.4.1. STANDARD ASSAY FOR EMULSIFYING ACTIVITY

In order to study the kinetics of bioemulsifier production byAcinetobacter Sp. ATCC 31012 and to compare the emulsifying activitiesof α-emulsans and β-emulsans, a series of simple sensitive assays forthese bioemulsifiers were developed. These assays were based upon thelarge increase in turbidity of a mixture of oil and water arising fromthe emulsion of the hydrocarbon in the aqueous phase.

The first assay involved the emulsification of gas-oil in sea waterunder standardized conditions and subsequent measurement of turbidity.When it was found that sea water could be replaced in the assayprocedure with dilute solutions of magnesium salts (cf/Section 8.4), asecond assay was developed involving emulsification of gas-oil in 10 mMof magnesium sulfate at pH 7.2. Finally, after it was found that thebioemulsifiers exhibited a degree of specificity toward differentclasses of hydrocarbon substrates (cf/Section 9), totally definedconditions were developed using a mixture of hexadecane and2-methylnaphthalene in place of gas-oil and buffered magnesium sulfate(or magnesium chloride) in place of sea water.

Each assay technique consisted of adding hydrocarbon (0.05 ml of gas-oilor 0.1 ml of 1:1 (v/v) hexadecane/2-methylnaphthalene) to 7.5 ml offiltered sea water or 7.5 ml of Tris-Mg buffer [20 mMtris-(hydroxymethyl)aminomethane hydrochloride, pH 7.2, supplementedwith 10 mM magnesium sulfate] containing 1 to 25 units of bioemulsifierper ml (about 3 to 75 mcg/ml of bioemulsifier) in a 125 ml flask. Afterreciprocal shaking (150 strokes per minute) for one hour at 26° C.,contents of the flask were transferred to Klett tubes for measurement ofturbidity in a Klett-Summerson colorimeter fitted with a green filter.Appropriate dilutions were made in water so that the final readings werebetween 30 and 150 Klett units, and values for Klett units reported asfinal readings times the dilution. Values for controls containing nobioemulsifier (5 to 20 Klett units) were subtracted. One unit ofbioemulsifier per ml is defined as that amount of activity which yields100 Klett units using 0.1 ml of 1:1 (v/v) hexadecane/2-methylnaphthaleneand 7.5 ml of Tris-Mg buffer. Specific Emulsification Activity (orspecific activity) is units per mg of bioemulsifier, dry weight basis.

FIG. 1 graphically illustrates standard curves obtained when all threeassay techniques were applied to an α-emulsan produced by growingAcinetobacter Sp. ATCC 31012 at 30° C. in a reciprocally shaken flask ona medium containing 1.0% (v/v) ethanol, 0.125% urea, 0.125% magnesiumsulfate [MgSO₄.7H₂ O], 0.0002% ferrous sulfate [FeSO₄.7H₂ O], 0.001%calcium chloride (anhyd), 0.025% dibasic potassium phosphate, and 0.2 MTris HCl buffer, pH 7.4. The preparation of α-emulsan used in preparingsuch curves had a Specific Emulsification Activity of 330 units per mg.Curve 1-A represents the relationship between the amount ofemulsification between 0.05 ml Gach-Saran gas-oil and 7.5 ml of filteredsea water; Curve 1-B represents the relationship between the amount ofemulsification between 0.05 ml Gach-Saran gas-oil and 7.5 ml Tris-Mgbuffer; and Curve 1-C represents the relationship between the amount ofemulsification between 0.1 ml 1:1 (v/v) hexadecane/2-methylnaphthaleneand 7.5 ml Tris-Mg buffer, all as a function of α-emulsan concentration.Each point in FIG. 1 represents the average of 3 to 4 determinations.These standard curves were then used to determine the emulsifyingactivity of preparations of crude and purified emulsans (α-emulsans,β-emulsans and the semi-synthetic ψ-emulsans) and apoemulsans(apo-α-emulsans, apo-β-emulsans and apo-ψ-emulsans). Characterization ofa particular Acinetobacter bioemulsifier as an α-emulsan or a β-emulsanis based on chemical analysis of the fatty acid esters contained in thelipoacyl portions of the protein-extracted lipopolysaccharides.

6.4.2. EXTRACELLULAR PRODUCTION OF α-EMULSANS

Measurement of extracellular emulsifying activity was determined atdifferent stages of growth of Acinetobacter Sp. ATCC 31012 in an ethanolmedium, the fermentation conditions being identical to those used toprepare the α-emulsan used for the standard assay tests. Growth wasestimated by turbidity using a Klett-Summerson colorimeter fitted with agreen filter or a Gilford Spectrophotometer (Model 240). One hundredKlett units of exponentially growing Acinetobacter Sp. ATCC 31012correspond to an absorbance at 620 nm (1-cm light path) of 0.816 and abiomass of 0.37 g per liter (dried at 90° C. for 16 hours).

FIG. 2 shows the relationship between the growth of Acinetobacter Sp.ATCC 31012 on the ethanol medium, the production of the bioemulsifier(α-emulsan) during such growth, and the change of pH during such growth,all as a function of time. Although these data are limited to theproduction of α-emulsan in a shaking flask fermentation with aparticular ethanol medium, FIG. 2 illustrates the general rule that theproduction of α-emulsan occurs during the growth phase.

6.4.3. EXTRACELLULAR PRODUCTION OF β-EMULSANS

Measurement of extracellular emulsifying activity was also determined atdifferent stages of growth of Acinetobacter Sp. ATCC 31012 in ahexadecane medium, the medium and fermentation conditions beingidentical to those used to prepare the emulsan used for the standardassay tests except that 0.2% (v/v) hexadecane medium was used in placeof ethanol as the carbon source. Viable cell number was determined byspreading 0.1 ml of an appropriate dilution on ACYE agar, whichcontained 0.5% sodium acetate, 0.1% yeast extract (Difco), 0.125% urea,0.025% dibasic potassium phosphate and 1.5% agar (Difco). Plates wereincubated at 32° C. for 3 days.

FIG. 3 shows the relationship between the growth of Acinetobacter Sp.ATCC 31012 on the hexadecane medium and the production of thebioemulsifier (β-emulsan) during such growth. The data contained in FIG.3 is similarly limited to the production of β-emulsan in a shaking flaskfermentation with a particular hexadecane medium, and shows that theproduction of β-emulsan also occurs during the growth period.

6.4.4. DISTRIBUTION OF EMULSIFYING ACTIVITY IN FRACTIONS OF GROWTHCULTURE

After 40 hours of incubation of Acinetobacter Sp. ATCC 31012 in theethanol medium and in the hexadecane medium as described above inSections 6.3.2 and 6.3.3, respectively, each culture was centrifuged at10,000×g for 15 minutes and the pellets washed once with Tris-Mg buffer.The pellicle formed during centrifugation of the hexadecane culture wasremoved, washed twice with growth medium before assaying for activity.Emulsifying activity in each fraction for the ethanol and hexadecanegrowth cultures was assayed by the standard assay technique describedabove in Section 6.4.1 and illustrated in FIG. 1. The results of suchassays are summarized in Table I.

                  TABLE I                                                         ______________________________________                                        Distribution of Emulsifying Activity in                                       Fractions of Growth Cultures                                                              Emulsifier (units/ml)                                                            Ethanol    Hexadecane                                          Fraction       Substrate  Substrate                                           ______________________________________                                        Pellet          7         0                                                   Supernatant fluid                                                                            23         14                                                  Pellicle       --         0                                                   ______________________________________                                    

The data contained in Table I show that over 75% of the activity wasextracellular when ethanol was the substrate, while at the measureableactivity was extracellular when Acinetobacter Sp. ATCC 31012 was grownon hexadecane medium. The small amount of activity associated with thepellet fraction was variable; in certain cases no measureable cell-boundactivity could be found. Disruption of the pellet fractions by sonicoscillation did not release additional emulsifying activity.

6.5. DEPROTEINIZATION

Apoemulsans may be prepared by deproteinization of the particularemulsans, which technique was used to isolate and purify samples for thechemical characterization of both Acinetobacter bioemulsifiers describedbelow. The associated protein may be separated from both bioemulsifiersby the hot phenol extraction technique described by O. Westphal et al.in the monograph edited by R. L. Whistler, entitled "CarbohydrateChemistry", Academic Press, Inc., New York, pp. 83-91. Alternatively,the protein fraction may be removed enzymatically by proteolyticdigestion.

6.6. ISOLATION AND PURIFICATION

The extracellular protein-associated lipopolysaccharides produced byAcinetobacter Sp. ATCC 31012 and their respective deproteinizedderivatives may be isolated and purified by various procedures,including selective precipitation, selective solvent extraction orpartitioning or selective adsorption onto a solid adsorbant followed bysubsequent elution or extraction. For many industrial uses, isolationand purification of the Acinetobacter bioemulsifiers is not necessary,since the cell-free growth media may be used directly. For the purposesof determining their respective structures as well as their chemical andphysical properties, particularly with respect to emulsifying activity,the α-emulsans and β-emulsans produced by Acinetobacter Sp. ATCC 31012have been isolated and purified. Three different procedures have beenfollowed, including (a) heptane partitioning of the crude extracellularlipopolysaccharide from the fermentation medium, followed by extractionof impurities from the heptane-partitioned biopolymer and subsequentwork-up; (b) precipitation of the extracellular lipopolysaccharide byammonium sulfate, followed by work-up of the precipitate; and (c)precipitation of the extracellular lipopolysaccharide by a detergentquaternary ammonium cation followed by work-up of the precipitate. Eachof these techniques is equally applicable to the isolation andpurification of the respective apoemulsans.

6.6.1. HEPTANE PARTITIONING

Because the Acinetobacter bioemulsifiers exhibit specificity withrespect to the structurally different types of hydrocarbon substrateswhich may be emulsified (cf/Section 9), certain water-immisciblehydrocarbons may be used to selectively extract the extracellularlipopolysaccharide from the fermentation media without creating a stableemulsion. By way of illustration, heptane extraction of the cell-freeculture medium from which ether-extractibles had been removed suspendedover 90% of extracellular lipopolysaccharide at the heptane/waterinterface. After evaporation of the heptane, and preferably furthersolvent extraction with ether, the resultant product is a viscous syrupwhich can be dissolved in 50% aqueous methanol, the impurities removedby dialysis and the remaining material recovered by lyophilization. In atypical example using this heptane partitioning technique, a purifiedβ-emulsan was prepared which was characterized by a specific activity of205 units per mg.

6.6.2. AMMONIUM SULFATE PRECIPITATION

The addition of ammonium sulfate to the fermentation broth has been usedto fractionally precipitate the extracellular lipopolysaccharides fromthe culture medium, from which the concentrate may be recovered andfurther treated to remove impurities. By way of illustration, additionof ammonium sulfate to cell-free supernatant fluids has resulted in theprecipitation of substantially all of the extracellularlipopolysaccharides when the concentration of ammonium sulfate isincreased from 30% saturation to a final concentration of 40%saturation. The resulting precipitate, which may be collected bycentrifugation, has been extracted by ether to remove impurities,dialyzed against water and lyophilized, yielding the purifiedextracellular lipopolysaccharide. In a typical example using thisammonium sulfate precipitation technique, a purified α-emulsan wasprepared which was characterized by a specific activity of 330 units permg.

6.6.3. QUATERNARY AMMONIUM PERCIPITATION

Because the extracellular lipopolysaccharides produced by AcinetobacterSp. ATCC 31012 were found to be anionic biopolymers, a procedure wasdeveloped to precipitate the anionic biopolymer with a cationicdetergent, such as cetyltrimethyl ammonium bromide, from whichprecipitate the detergent cation could be separated while leaving thepurified extracellular lipopolysaccharide. For example, the addition ofcetyltrimethyl ammonium bromide to an aqueous solution of α-emulsanimmediately forms a precipitate which is recoverable by centrifugationor filtration. This precipitate is soluble in 0.1 M sodium sulfate, fromwhich solution cetyltrimethyl ammonium iodide precipitates upon additionof potassium iodide, leaving the α-emulsan in the supernatant fluid.Dialysis of this supernatant fluid against distilled water, followed bylyophilization, has yielded highly purified samples of α-emulsan as awhite solid, with a specific activity of 350 units per mg.

7. CHEMICAL AND PHYSICAL PROPERTIES OF EMULSANS AND APOEMULSANS

Chemical and physical characterization of emulsans and apoemulsans weremeasured on samples which had been purified to apparent homogeneity,from which characterization conclusions were reached on the structure ofthese unique extracellular lipopolysaccharides. Such information isnecessary to give a better understanding of the relationship between themolecular structure of this class of bioemulsifiers and theirspecificity in emulsifying various hydrocarbon substrates.

7.1. PREPARATION OF SAMPLES FOR ANALYTICAL CHARACTERIZATION 7.1.1.PREPARATION OF EMULSAN SAMPLES

The emulsan samples used for chemical and physical characterization wereprepared by aerobically growing Acinetobacter Sp. ATCC 31012 on anethanol medium (α-emulsan) or a hexadecane medium (β-emulsan) and werepurified by precipitation between 30-40% ammonium sulfate saturation,followed by extraction with ether, dialysis against distilled water andlyophilization, as described more fully in the example set forth belowin Section 13.7. Some samples of α-emulsan were further purified byemploying the cetyltrimethyl ammonium bromide precipitation technique,as described more fully in the example set forth below in Section 13.11.

7.1.2. PREPARATION OF APOEMULSAN SAMPLES

The apoemulsan samples used for chemical and physical characterizationwere prepared by hot phenol extraction of the associated protein moietyfrom the emulsan samples. The deproteinization procedure, which isdescribed more fully in the examples set forth below in Sections 13.6and 13.7, involved adding a dilute solution (5 mg/ml) of emulsanpreheated to 65°-68° C. to an equal volume of 90% phenol at 65° C.,stirring the mixture for 15 minutes while maintaining the temperature at65° C., and then cooling the mixture to 10° C. in an ice bath. Theresulting emulsion was then centrifuged to separate the denaturedprotein in the phenol phase from the apoemulsan in the aqueous phase.After transferring the viscous aqueous phase to a flask, the phenollayer and phenol/water interface were extracted three more times withwater, following which the combined water extracts were dialyzedextensively against several changes of distilled water and thenfreeze-dried, yielding 85% by weight of apoemulsan based on the weightof the emulsan. All of the emulsifying activity was in the recoveredemulsan. None of the emulsifying activity was in the denatured proteinfraction.

7.1.3. AMMONIUM SULFATE FRACTIONATION OF APO-α-EMULSAN

To assure homogeneity of the apo-α-emulsan, the deproteinizationprocedure was repeated on another sample of α-emulsan which had beemprepared by aerobically growing Acinetobacter Sp. ATCC 31012 on anethanol medium and which had been purified by precipitation between30-40% ammonium sulfate fractionation, followed by extraction withether, dialysis against distilled water and lyophilization. After threephenol extractions, the combined water extracts were extracted fourtimes with an equal volume of ether to remove residual phenol. Followingevaporation of any retained ether, the viscous aqueous phase was cooledto 5° C. and brought to 32.5% ammonium sulfate saturation. Afterstanding for one hour at 5° C., the clear translucent precipitate wascollected at centrifugation at 5,000×g for 30 minutes at 5° C. Theprocedure was repeated to obtain a slightly turbid second precipitatebetween 32.5% and 35% ammonium sulfate saturation and another smallprecipitate between 35% and 40% ammonium sulfate saturation. Noadditional precipitate formed between 40% and 60% saturation.

Each of the precipitates was dissolved in water and was then dialyzed at2°-5° C. successively against distilled water, 0.05 N hydrochloric acidfor 24 hours and double distilled water, following which each of theresulting solutions were freeze-dried. Over 99% of the emulsifyingactivity of the apo-α-emulsan was found in the two fractions whichprecipitated between 30% and 35% ammonium sulfate saturation. These twofractions contained similar specific activities and exhibitedsubstantially the same chemical composition. In addition, both fractionswere homogeneous when examined by immunodiffusion against antibodiesprepared against β-emulsan, each giving a single identical band uponOuchterlony two-dimensional diffusion. Accordingly, the two fractionswere combined for certain of the chemical and physicalcharacterizations, the combined fractions when used being identifiedherein as "apo-α-emulsan-WA".

7.1.4. QUATERNARY AMMONIUM SALT PRECIPITATION OF APO-α-EMULSAN

To cross-check the analytical data on apo-α-emulsan-WA, another highlypurified sample of apo-α-emulsan was prepared using (1) the identicalhot phenol extraction of another sample of α-emulsan which had beenprepared by aerobically growing Acinetobacter Sp. ATCC 31012 on anethanol medium, followed by (2) cetyltrimethyl ammonium bromideprecipitation of the resultant apo-α-emulsan, dissolving the precipitatein 0.1 M sodium sulfate, and addition of potassium iodide to thesolution to precipitate cetyltrimethyl ammonium iodide. The supernatantfluid contained the apo-α-emulsan. Extensive dialysis of thissupernatant fluid against distilled water followed by lyophilizationyielded a highly purified apo-α-emulsan which was designated as"apo-α-emulsan-CTAB".

7.2. CHEMICAL CHARACTERIZATION 7.2.1. CHEMICAL COMPOSITION OF EMULSANSAND APOEMULSANS

Elemental analyses of α-emulsan and apo-α-emulsan, which were performedon samples of α-emulsan and apo-α-emulsan-WA that had been dried toconstant weight at 55° C. in vacuo (the apo-α-emulsan-WA sample havingreleased 12.7% water on such drying), are shown in Table II.

                  TABLE II                                                        ______________________________________                                        Elemental Composition of Emulsan                                              Sample       % C    % H      % N  % S    % Ash                                ______________________________________                                        α-Emulsan                                                                            41.72  6.95     7.74 0.7    13.8                                 Apo-α-emulsan-WA                                                                     46.70  7.01     6.06 0.0     3.5                                 ______________________________________                                    

The deproteinized sample (apo-α-emulsan-WA) contained significantly lessN, S and ash then emulsan. The C:N:H ratio of apo-α-emulsan-WA wascalculated to be 9.0:1.0:16.1. No significant quantities (<0.5%) ofphosphorous or halides were found in either sample. Functional grouptests were positive for carboxyl and ester groups and negative formethoxy and ethoxy groups. The polymer contained less than 0.02micromoles reducing sugar per mg, which was the sensitivity of the testemployed. The nonreducing polymer was resistant to high temperatures inneutral and alkaline conditions. No emulsifying activity was lost at100° C. for 2 hours in distilled water; 50% of the activity remainedeven after treatment in 1 N sodium hydroxide at 100° C. for 1 hour.Apo-α-emulsan-WA was considerably more sensitive to acid, losing 50% ofits emulsifying activity in 2 minutes at 100° C. in 1 N hydrochloricacid.

Titration of apo-α-emulsan-WA (40 mg/4 ml) between pH 2.5-10.5 showed asingle inflection point, corresponding to pK'=3.05 (identical to astandard sample of glucuronic acid). Apo-α-emulsan-WA consumed 0.24micromoles periodate per mg, (which would suggest the presence of asmall amount of glucose in the polymer), which was subsequentlydetermined to be due to a small amount of glucose present in an ammoniumsulfate co-precipitated extracellular polysaccharide which possessed noemulsifying activity. Periodate uptake ceased after two hours at 30° C.,pH 4.5. The periodate treated material did not lose any emulsifyingactivity, further indicating that no glucose was present in theapo-α-emulsan.

7.2.2. ALKALINE HYDROLYSIS OF APOEMULSAN

Two hundred milligrams of apo-α-emulsan-WA were refluxed in 40 ml of 1 Nsodium hydroxide for 4 hours, cooled, extracted three times with 40 mlether, acidified to pH 1-2 with concentrated hydrochloric acid, andextracted again three times with 40 ml ether. The acid-ether extractswere combined and dried in a tared flask, yielding 30 mg (15%) fattyacids; extraction with ether prior to acidification yielded less than 2mg dry material. Combining the weight recovery of fatty acid from thepolymer (150 mcg/mg) and the 0-ester content (0.65 micromoles/mg) yieldsan average equivalent weight of 231 for the fatty acids.

7.2.3. ACID HYDROLYSIS OF APOEMULSAN

Preliminary hydrolysis studies were performed on apo-α-emulsan at 80° C.and 100° C. in sealed tubes with concentrations of hydrochloric acidvarying from 0.01-6.0 M. After removal of hydrogen chloride in vacuo,the products were examined for reducing power, amino sugars and by paperchromatography in n-butanol/pyridine/water (6:4:3, v/v) [Solvent A] andin n-propanol/ethyl acetate/water (7:1:2, v/v) [Solvent B].

FIG. 4 is a graphical representation of the changes which occur on acidhydrolysis of apo-α-emulsan. The weight percent of reducing power isplotted against the duration of hydrolysis at 100° C. at 0.05 M HCl(shown in the lower curve) at 5 M HCl (shown in the upper curve).Hydrolyses were performed in sealed tubes under nitrogen on 1 mg/mlsamples of apo-α-emulsan. As shown in FIG. 4, at 0.05 M hydrochloricacid at 100° C. there was a release of around 6% reducing sugar duringthe first hour, followed by a slower release of about 1% reducing sugarper hour for the next 20 hours.

After 27-hour hydrolysis in 0.05 M HCl at 100° C., chromatographyrevealed the presence of two major reducing spots (subsequentlyidentified as galactosamine and an aminouronic acid) and one minorcomponent (subsequently identified as glucose). [N.B.-Analytical workdone much later on CTAB-fractionated material indicates that thepresence of glucose was due to an impurity which was co-precipitatedduring the ammonium sulfate fractionation of apo-α-emulsan.] Inaddition, there were considerable amounts of incompletely hydrolyzedmaterial (remaining near the origin). After 5-hour hydrolysis in 0.05 MHCl, only glucose was detected on the chromatograms. N-acetylatedderivitives of the amino sugars were never detected.

Maximum amount of reducing sugar was obtained by hydrolyzingapo-α-emulsan in 5 M HCl at 100° C. for 30 minutes. Even under theseconditions significant amounts of emulsifying agent were incompletelyhydrolyzed. Longer periods of hydrolysis resulted in further destructionof the sugars. The relative amount of amino sugars to glucose increasedwith time of hydrolysis due both to the slower release of amino sugarsfrom the polymer and faster destruction of free glucose. Hydrolysis ofsamples of the ammonium sulfate fractionated apo-α-emulsan-WA showed thesame chromatograhic pattern as that of apo-α-emulsan; however, when thisanalysis was repeated on the sugars produced by hydrolysis ofapo-α-emulsan-CTAB at 100° C. in 0.05 N and 5 N HCl for the same periodsof time, no glucose was detected. Following hydrolysis in 5 M HCl at100° C. for 30 minutes, apo-α-emulsan-WA released 37.6% reducing sugarand 24.4% total hexosamines (in both cases, using galactosamine as thestandard).

7.2.4. IDENTIFICATION OF SUGAR COMPONENTS

Table III summarizes the data that led to the conclusion that the sugarsproduced by hydrolysis of ammonium sulfate fractionated apo-α-emulsanwere D-glucose (minor), D-galactosamine (major) and an aminouronic acid(major). Unknown compound A did not separate from glucose in solvents Aor B and yielded a positive D-glucose reaction directly on the paper.Unknown compound B migrated identically to galactosamine in solvent B,gave a positive D-galactose oxidase reaction and was converted to lyxose(R_(Glc) =1.49 in solvent B) by ninhydrin degradation. Unknown compoundC gave positive reactions for reducing sugar, amino sugar andcarboxylate ion. Moreover, it was similar both in chromatographicbehavior and in its reaction with the nitrous acid-indole test to2-amino-2-deoxyhexuronic acids.

                  TABLE III                                                       ______________________________________                                        Properties of Sugar Products                                                  of Hydrolysis of Ammonium                                                     Sulfate Fractionated Apo-α-Emulsan                                      Component.sup.a   R.sub.Glc.sup.b                                                                      Positive reactions.sup.c                             ______________________________________                                        Standards:                                                                    D-glucose         1.25   glucose oxidase                                      D-galactose       1.22   galactose oxidase                                    D-glucosamine     1.00   ninhydrin (purple),                                                           EM, glucose oxidase                                  D-galactosamine   0.85   ninhydrin (purple),                                                           EM, galactose oxidase                                D-N--acetylgalactosamine                                                                        1.58   EM                                                   Acid hydrolysis products of                                                   apo-α-emulsan:                                                            A               1.25   glucose oxidase                                        B               0.85   ninhydrin (purple), EM                                                        galactose oxidase                                      C               0.23   ninhydrin (greenish-                                                          yellow, later blue), EM                              ______________________________________                                         .sup.a Obtained after 24 hour hydrolysis of apoα-emulsan in 0.05 M      HCl at 100° C.                                                         .sup.b Rate of movement of each sugar relative to glucosamine in solvent      A.                                                                            .sup.c All components gave positive alkaline silver nitrate tests. Spot       tests were determined directly on the chromatograms. EM is the modified       Elson and Morgan reagent [R.W. Wheat in the monograph edited by E. F.         Neufeld et al., "Methods in Enzymology", Vol. VIII, Academic Press Inc.,      New York, pp. 60-78.                                                     

Based on all the evidence, therefore, it is certain that the polymer ispoly[D-galactosamine/aminouronic acid]. Any glucose present is probablyan impurity.

7.2.5. IDENTIFICATION OF FATTY ACIDS

As a general rule, the esterified fatty acid content of apo-α-emulsansderived from the deproteinization of α-emulsans prepared by aerobicallygrowing Acinetobacter Sp. ATCC 31012 on an ethanol medium is in therange from about 7% to about 15%, corresponding to about 0.3 to about0.7 micromoles per milligram of fatty acid esters in which the fattyacids have an average equivalent weight from about 200 to about 230.Alkaline hydrolysis, acidification and ether extraction of α-emulsanyields a mixture of fatty acids, the infrared spectrum of whichexhibited absorption peaks at 3610 cm⁻¹ (nonbonded O--H), 3500 cm⁻¹(bonded O--H), 1705 cm⁻¹ (C═O) and 1050 cm⁻¹ (C--OH). The NMR spectrumin CDCl₃ indicated that the mixture consisted mainly of saturated andhydroxy-substituted fatty acids.

Base hydrolysis of one gram of α-emulsan was performed in 400 ml of 2.5%potassium hydroxide in 90% methanol under reflux for 4 hours. Afterremoval of the methanol in vacuo, 500 ml of water were added. The clearalkaline solution was washed three times with 150 ml of ether, the etherdiscarded, and the aqueous solution acidified to pH 2 with hydrochloricacid. The acid solution was then extracted five times with 100 ml ether,the interphase in each extraction being set aside. The combinedinterphase fractions were treated with acetone to precipitate proteinand polysaccharide. After removal of the precipitate by filtration andthe acetone by distillation in vacuo, the aqueous phase was againextracted with ether. The combined ether extracts were dried overmagnesium sulfate. Removal of the ether left 130 mg (13% yield) of amixture of fatty acids. The methyl esters of the fatty acid mixture wereprepared with diazomethane by standard techniques.

Gas liquid chromatography of the methyl esters of the fatty acid mixtureled to the separation of eleven peaks, nine of which were identified bycomparison of retention volumes of pure samples of known structure.Table IV sets forth the relative retention volumes of the methyl estersof the fatty acids obtained from emulsan.

                  TABLE IV                                                        ______________________________________                                        Fatty Acid Methyl Esters                                                      Obtained from Mild Base                                                       Hydrolysis of χ-Emulsan                                                                              Relative                                                       Fatty Acid     Retention                                          Peak No.    Methyl Ester   Volume                                             ______________________________________                                        1           Decanoic       0.17                                               2           Dodecanoic     0.29                                               3           Dodecenoic     0.34                                               4           Unidentified   0.48                                               5           Unidentified   0.61                                               6           Hexadecanoic   1.00                                               7           Hexadecenoic   1.14                                               8           2-Hydroxydodecanoic                                                                          1.30                                               9           3-Hydroxydodecanoic                                                                          1.69                                               10          Octadecanoic   1.94                                               11          Octadecenoic   2.16                                               ______________________________________                                    

Although the relative amounts of fatty acids will vary from sample tosample, in general, the two hydroxydodecanoic acids comprise from about50% to about 70% of the aggregate fatty acids, with 3-hydroxydodecanoicacid usually predominating over 2-hydroxydodecanoic acid. Table V setsforth the fatty acid composition of the α-emulsan described above.

                  TABLE V                                                         ______________________________________                                        Typical Fatty Acid                                                            Ester Composition of α-Emulsan                                                          Percent of Total                                              Fatty Acid      Fatty Acids                                                   ______________________________________                                        Decanoic        11.4                                                          Dodecanoic      23.0                                                          Dodecenoic       2.4                                                          2-Hydroxydodecanoic                                                                           10.5                                                          3-Hydroxydodecanoic                                                                           39.5                                                          Hexadecanoic     0.7                                                          Hexadecenoic    trace                                                         Octadecanoic     0.3                                                          Octadecenoic    trace                                                         Unidentified    12.0                                                          ______________________________________                                    

The acetone-precipitated polysaccharide remaining after 0-deacylation ofthe α-emulsan by mild base hydrolysis was redissolved in water, dialyzedextensively against water, lyophilized and then subjected to acidhydrolysis for 6 hours at 98° C. in 5 M HCl. The aqueous hydrolysate wasextracted with ether and the ether extract was treated by diazomethaneto convert to methyl esters whatever fatty acids remained after suchstrong acid hydrolysis. Gas chromatographic analysis of this materialrevealed the presence of methyl 3-hydroxydodecanoate as the only fattyacid. This showed that N-(3-hydroxydodecanoyl) groups were also presentin ψ-emulsan.

7.3. PHYSICAL CHARACTERIZATION

Preliminary experiments indicated that the purified α-emulsan wasexcluded by Sephadex G-100 and G-200 and did not pass an Amicon XM-30filter. This data, coupled with the fact that apo-α-emulsan contained1.5 micromoles of carboxylic groups per mg, suggested that thelipopolysaccharide was an anionic polymer. Additional data on physicalcharacterization is set forth below:

7.3.1. INTRINSIC AND REDUCED VISCOSITY

The intrinsic viscosities of the analytical samples of α-emulsan,apo-α-emulsan and apo-α-emulsan-WA in 0.15 M Tris buffer, pH 7.4, were470, 505 and 750 cc per gm, respectively. With all three samples,reduced viscosity was independent of concentration between 0.05 and 1.0mg per ml. Exposure of 0.5 mg per ml apo-α-emulsan to sonic oscillations(Branson B12 sonifier, setting 8, 14 min) reduced the reduced viscosityto 420 cc per gm. Exposure for an additional 20 minutes did not furtherreduce the viscosity. The viscosity of apo-α-emulsan as a function ofionic strength is shown in FIG. 5. Between 0.03-0.15 M NaCl, reducedviscosity decreased slightly from 515 to 480 cc per gm. The largeincrease in reduced viscosity at low ionic strengths is characteristicof polyelectrolytes and has been attributed to dilution of counterions.Reduced viscosity was also measured as a function of pH using 0.05 Mcitrate-phosphate buffer (pH 3-7) and 0.05 M Tris HCl buffer (pH6.8-8.5). Throughout the entire range (pH 3-8.5) the reduced viscosityof α-emulsan remained at 480±50 cc per gm.

7.3.2. SEDIMENTATION VELOCITY ANALYSIS

Sedimentation velocity analysis of 2 mg/ml of apo-α-emulsan-WA in 0.15 MNaCl showed a single broad band corresponding to an s₂₀ =6.06×10⁻¹³ secor 6.06 S. The diffusion coefficient, D, also determined in theanalytical centrifuge was 5.25×10⁻⁸ cm² sec⁻¹. The partial specificvolume of the material, V, was 0.712 cm³ g⁻¹.

7.3.3. ESTIMATION OF MOLECULAR WEIGHT

Estimating the molecular weight of apo-α-emulsan-WA from the equation,M=RTs/D(1-Vρ), where R is the gas constant, T is the absolutetemperature and ρ is the density of the solution, yields a weightaverage molecular weight of 9.76×10⁵. Alternatively, the molecularweight can be estimated using the determined values for intrinsicviscosity, η, sedimentation constant, S, and partial molar volume, V,according to the equation of Scheraga and Mandelkern [J. Am. Chem. Soc.,75, 179 (1953)]. The calculated viscosity average molecular weight forapo-α-emulsan-WA was 9.88×10⁵.

7.3.4. SPECTRAL PROPERTIES

The ultraviolet absorption spectrum of apo-α-emulsan-WA (220-350 nm)showed no maxima. The infrared spectrum of apo-α-emulsan incorporatedinto a KBr pellet or nugol revealed the following groups: 3340 cm⁻¹(O--H), 2900 cm⁻¹ (C--H), 1720 cm⁻¹, weak (C═O), 1640 cm¹ (amide I) and1545 cm⁻¹ (amide II). X-ray diffraction analysis of apo-α-emulsan, whichwas performed on a film formed by evaporation of a water solution ofapoemulsan, showed crystallinity. Table VI summarizes the 2θ angles andd spacings measured for the X-ray diffraction pattern recorded with Nifiltered CuKα radiation.

                  TABLE VI                                                        ______________________________________                                        X-Ray Diffraction Analysis of Apo-α-emulsan                             2θ°                                                                              d(A)   I(rel.)                                                ______________________________________                                        21.00           4.23   S                                                      16.70           5.31   W                                                      14.80           5.99   VW                                                     13.04           6.79   W                                                      10.66           8.30   W                                                      7.18            12.30  S                                                      ______________________________________                                    

7.4. CONCLUSIONS ON STRUCTURE

The foregoing data show that apo-α-emulsan is a highly acidiclipopolysaccharide with a molecular weight average close to one million.Molecular weight determination from sedimentation and diffusion dataclosely fit the value obtained from a consideration of sedimentation andviscosity measurements. In both cases the determined value for thepartial molar volume of 0.712 cm³ gm⁻¹ was used. The relatively highintrinsic viscosity, low diffusion constant and low sedimentationcoefficient of the emulsifier indicate that the shape of apo-α-emulsanis highly asymmetrical. Using Simha's factor [C. Tanford, "PhysicalChemistry of Macromolecules", John Wiley and Sons, Inc., New York, 1963,pp. 390-411] for the viscosity increment of rod-shaped ellipsoidsindicates that apo-α-emulsan has an axial ratio of close to 100.Preliminary examination of the purified apo-α-emulsan by electronmicroscopy revealed thin fibers with lengths greater than 1000 A.

Apo-α-emulsan is composed of major amounts of two amino sugars(D-galactosamine and an aminouronic acid) and a mixture of fatty acidesters in which the fatty acids (a) contain from 10 to 18 carbon atoms,and (b) possess an average equivalent weight from about 200 to about230, about 50% or more of such fatty acids being composed of2-hydroxydodecanoic acid and 3-hydroxydodecanoic acid, with the latterhydroxy fatty acid predominating.

Titration curves and infrared spectrum of the apo-α-emulsan sampleindicate that the amino sugars of the biopolymer are N-acylated. Theaminouronic acid content of the apo-α-emulsan sample was estimated byacid-base tritration of the biopolymer to be 1.5 micromoles/mg. Assumingthe aminouronic acid to be an N-acetylhexosamine uronic acid (M.W.=222),it would comprise 33% by weight of the biopolymer. Direct estimation ofD-galactosamine content of the apo-α-emulsan sample is not possible atthis time, since hydrolysis conditions necessary to release it fromapoemulsan cause considerable decomposition of the amino sugar. Roughestimates (from intensities of reducing and ninhydrin positive materialson chromatograms) indicate that the amount of D-galactosamine is similarto the quantity of aminouronic acid. The total fatty acid ester contentof the apo-α-emulsan sample was 15% by weight with an average equivalentweight of about 231. Table VII summarizes the chemical composition ofapo-α-emulsan-WA on the basis of all the data.

                  TABLE VII                                                       ______________________________________                                        Chemical Composition of Apo-α-emulsan-WA                                Component        Apo-α-Emulsan-WA                                       ______________________________________                                        D-galactosamine.sup.a                                                                          20-30.sup.b                                                  Hexosamine uronic acid.sup.a                                                                   33.3                                                         D-glucose.sup.c  5.2                                                          Fatty acid esters.sup.d                                                                        15.0                                                         Water            12.7                                                         Ash              3.5                                                          ______________________________________                                         .sup.a Calculated as N--acetylated amino sugar.                               .sup.b Estimated from intensity of ninhydrin and reducing spots on            chromatograms.                                                                .sup.c Probably present as an impurity in apoα-emulsan-WA.              .sup.d See TABLE V for typical fatty acid distribution.                  

7.5. VARIATIONS IN STRUCTURE

Table VII summarizes the chemical composition of apo-α-emulsan-WA, whichis a highly purified sample free of protein and nucleic acid and whichappeared to be homogeneous by several criteria, namely (a) only a singleband was found by Ouchterlony two-dimensional diffusion; (b) only asingle component was observed by sedimentation velocity studies, usingseveral concentrations of material; and (c) attempts to further purifythe material by extraction or precipitation with organic solvents didnot improve its specific activity or alter its chemical composition.

Growth of Acinetobacter Sp. ATCC 31012 on a utilizable carbon source(such as ethanol, palmitic acid or dodecane) to produce thosebioemulsifiers which are characterized as α-emulsans will yield productsin which the 0-lipoacylated heteropolysaccharide may deviate from thespecific chemical composition for apo-α-emulsan-WA shown in Table VII.As a general rule, the N-acyl and partially 0-acyl heteropolysaccharidesin the emulsan or constituting the apo-α-emulsan will be composed on adry weight basis of from about 20% to about 35% by weight ofD-galactosamine; from about 30% to about 35% by weight of hexosamineuronic acid; and from about 7% to about 19% by weight of fatty acidesters in which the fatty acids contain from about 10 to about 18 carbonatoms and are characterized by an average equivalent weight from about200 to about 230, from about 50% to about 70% of such fatty acids in the0-lipoacyl portion of the apo-α-emulsan being composed of2-hydroxydodecanoic acid and 3-hydroxydodecanoic acid. Although theratio of 2-hydroxydodecanoic acid to 3-hydroxydodecanoic acid in the0-lipoacyl portion of the apo-α-emulsan (or apo-α-emulsan component ifthe product is an α-emulsan) may vary from about 1:4 to about 1:1, the3-hydroxydodecanoic acid will predominate in those biopolymers whichhave a high Specific Emulsification Activity.

7.6. IMMUNOLOGICAL CHARACTERIZATION

To immunologically characterize the Acinetobacter bioemulsifiersproduced by Acinetobacter Sp. ATCC 31012, rabbits were injected with 1mg of β-emulsan in 1 ml complete Freund adjuvant. The rabbits were bled11 to 14 days later, from which sera a crude immunoglobulin fraction wasobtained by ammonium sulfate fractionation.

Antibodies prepared against β-emulsan cross-react in an identicalfashion with α-emulsan, apo-α-emulsan, apo-β-emulsan, ψ-emulsan(produced by mild base hydrolysis of α- or β-emulsan) and proemulsan(produced by strong base hydrolysis of any of the foregoing), indicatingthat both Acinetobacter bioemulsifiers (α-emulsan and β-emulsan) andtheir various deproteinated and deacylated derivatives haveapproximately the same polymer backbone, even though these classes ofbiopolymers are distinguishable by fatty acid ester content as well asby differences in the distributions of fatty acids, the α-emulsanscontaining a larger amount and greater proportion of 3-hydroxydodecanoicacid ester than the β-emulsans.

8. EMULSIFYING PROPERTIES

Data are presented below with respect to the emulsifying properties ofboth types of extracellular lipopolysaccharides (α-emulsans andβ-emulsans) produced by Acinetobacter Sp. ATCC 31012 so thatsimilarities as well as differences between these biopolymers may beunderstood. As before, unless the particular type of extracellularlipopolysaccharide produced by the organism is identified by name, thephrase "Acinetobacter bioemulsifier" refers collectively to both classesof emulsans. Unless otherwise indicated, emulsifying activity wasassayed in accordance with the standard assay technique described abovein Section 6.4.1 using the standard curves shown in FIG. 1.

8.1. KINETICS OF EMULSAN-INDUCED EMULSION FORMATION

The rate of emulsification of gas-oil by purified Acinetobacterbioemulsifier is summarized in FIG. 6, in which the numbers identifyingeach curve refer to the weight ratios of gas-oil/bioemulsifier. At fixedconcentrations of bioemulsifier (0.25 mg in FIG. 6A and 0.7 mg in FIG.6B, each in 7.5 ml of filtered sea water), using amounts of gas-oilvarying from 4.5 to 582 mg and under the conditions (i.e., reciprocalshaking at 150 strokes per minute for 1 hour at 25° C. ) of the standardassay technique, the rate of emulsion formation as well as the finalturbidity were proportional to gas-oil concentration between 5 to 100 mgof gas-oil per ml. With 33 or 100 mcg/ml of bioemulsifier andconcentrations of gas-oil exceeding 45 mg/ml, half-maximum turbiditieswere reached in less than 5 minutes. When the bioemulsifier and gas-oilwere allowed to interact at 25° C. for 2 hours without shaking,half-maximum turbidities were obtained in less than 2 minutes ofshaking. After 60 minutes of shaking, turbidity continued to increasegradually for 4 hours at about 10% per hour.

Emulsion formation as a function of gas-oil concentration is shown inFIG. 7, in which the lower curve represents the data obtained using 33mcg/ml of bioemulsifier and the upper curve the data obtained using 100mcg/ml of bioemulsifier, both in filtered sea water, with varyingamounts of gas-oil. Each mixture was reciprocally shaken for 60 minutesat 150 strokes per minute, and emulsion formation then measured.Emulsions were formed over the entire gas-oil concentration rangestudied, 0.5 to 100 mg per ml. Below 1.5 mg gas-oil per ml, turbiditieswere directly proportional to gas-oil concentration. Between 8 to 30 mggas-oil per ml, turbidity increased about 5 Klett units per mg gas-oil.

8.2. EFFET OF pH AND SALT CONCENTRATION ON EMULSION FORMATION

A cinetobacter bioemulsifier-induced emulsification of gas-oil as afunction of pH is shown in FIG. 8. The data shown in FIG. 8 were basedon reciprocally shaking (150 strokes per minute at 25° C. for 60minutes) flasks which contained 33 mcg/ml of bioemulsifier and 6 mg/mlof Agha-Jari gas-oil in 7.5 ml of either (a) sea water [closed circles];(b) 10 mM NaCl [open circles]; (c) 100 mM citrate-phosphate buffer[triangles]; or (d) 50 mM Tris-NaOH buffer [squares]. The pH of seawater and 10 mM NaOH were adjusted by addition of HCl or NaOH.

In sea water, near maximum emulsions were obtained from pH 5 to at leastpH 9. Above pH 9 precipitation of salts prevented accurate measurementsof emulsion. In aqueous solutions containing Tris buffer,citrate-phosphate buffer, or diluted saline, a sharp maximum wasobtained between pH 5-6. Above pH 7, activity was completely lost.

In order better to understand the different results obtained in seawater and fresh water, the effect of salts on bioemulsifier-inducedemulsification was measured at pH 7.0 and the data summarized in FIG. 9.The data shown in FIG. 9 was based on the emulsification of gas-oil withthe Acinetobacter bioemulsifier in distilled water to which had beenadded varying concentrations of magnesium chloride (closed circles) orsodium chloride (open circles). Emulsification was measured afterreciprocally shaking (150 strokes per minute) the flasks for 60 minutesat 25° C.

Maximum activity was obtained with 5-40 mM magnesium sulfate ormagnesium chloride. Half maximum activity was achieved with 1.5 mMmagnesium ions (Mg⁺⁺). Calcium chloride (10 mM) and manganese chloride(10 mM) could be substituted for magnesium sulfate. On the other hand,sodium chloride (10-500 mM) had little effect on emulsion formation,either in the presence or absence of magnesium ions. Consequently, theability of Acinetobacter bioemulsifiers to emulsify hydrocarbons abovepH 6 is dependent upon divalent cations and appears to be independent ofsodium chloride conentration. Because of this property, thesebioemulsifiers are capable of functioning in the presence of highconcentrations of sodium chloride found in sea water or connate water.

8.3. STABILITY OF BIOEMULSIFIER-INDUCED EMULSIONS

Gas-oil emulsions formed in the presence of Acinetobacter bioemulsifierslowly separate into two phases when allowed to stand undisturbed;namely, a lower clear aqueous phase and a turbid upper phase containingconcentrated oil droplets, bound bioemulsifier and water. As observedwith a phase microscope, emulsion breakage (demulsification) was aresult of "creaming" due to density differences between the two phasesand was not accompanied by droplet coalescence or aggregation. The rateof phase separation was followed by turbidity measurements in a Kletttube to determine the stability of the emulsion as a function of theratio gas-oil/bioemulsifier, the results being summarized in FIG. 10.Emulsions were formed after 120 minutes at 25° C. by reciprocallyshaking varying concentrations of gas-oil with either 33 mcg/ml (FIG.10A) or 100 mcg/ml (FIG. 10B) of Acinetobacter bioemulsifier, and thenallowed to stand without shaking from zero time (i.e., immediately afterformation of the emulsion) until 120 minutes. In FIGS. 10A and 10B,percent Klett units (Klett units at t=x divided by Klett units at t=O,expressed as percentage) are plotted against standing time. The numberson each curve refer to the weight ratios of gas-oil/bioemulsifier.

As shown in FIGS. 10A and 10B, emulsion stability depended more upon theratio of gas-oil/bioemulsifier than on the absolute concentration ofbioemulsifier or gas-oil used to form the emulsion. Withgas-oil/bioemulsifier ratios of less than 25, over 24 hours standing wasrequired for a 50% decrease in turbidity. With ratios between 25-200 and200-1000, half-maximum turbidities were reached in 1-24 hours and 10-60minutes, respectively. In all cases, the upper "cream" immediatelydispersed in aqueous media. Emulsion breakage was enhanced by divalentcations.

Rate of floatation of oil droplets as a function ofgas-oil/bioemulsifier ratio is shown in FIG. 11, in which the uppercurve represents data obtained using 100 mcg/ml of bioemulsifier and thelower curve represents data obtained using 33 mcg/ml of bioemulsifier,both with different gas-oil concentrations. The average radii of thedroplets, r, were calculated from Stokes equation V=21800 r², where V isthe velocity at which oil droplets rise in cm/sec and r is the radius incm, using 0.90 g cm⁻³ as the density of gas-oil. The calculated dropletsizes were in good agreement with measurement of droplet size by phasemicroscopy (using a calibrated eye-piece micrometer). With a ratio ofgas-oil/bioemulsifier of 50, the droplets were barely visible by lightmicroscopy.

8.4. LOWERING OF OIL/WATER INTERFACIAL TENSIONS

The ability of Acinetobacter bioemulsifiers to lower the interfacialtensions between a series of n-alkanes and sea water is shown in FIG.12, which illustrates the interfacial tensions of n-alkanes from 6 to 16carbon atoms in sea water containing 0.1% bioemulsifier. Values forinterfacial tension were determined at 27° C. using the spinning dropinterfacial tensiometer. Using similar techniques, the interfacialtensions between Prudhoe Bay crude oil and sea water were measured using1 and 10 mg of bioemulsifier per ml, yielding 8.3 and 6.9 dynes per cm,respectively.

9. SPECIFICITY OF THE HYDROCARBON SUBSTRATE

Apart from classification as anionic, cationic or nonionic, mostemulsifiers are described in terms of their HLB numbers, which is ameasure of the hydrophile-lipophile balance of the emulsifier. Veryoften, emulsifiers with similar HLB numbers interact differently withhydrocarbon substrates. Because biologically produced polymers oftenexhibit specificities not found in chemically synthesized materials, thehydrocarbon substrate specificity for Acinetobacterbioemulsifier-induced emulsion formation was studied using a widevariety of pure hydrocarbons, binary mixtures of hydrocarbons, crudeoils, fractions of crude oils and mixtures of crude oil fractions andpure hydrocarbons.

9.1. EMULSIFICATION OF PETROLEUM FRACTIONS

The ability of α-emulsans and β-emulsans to emulsify crude oil andfractions of crude oil is summarized below in Table XIII. All crude oilstested were emulsified by both types of Acinetobacter bioemulsifiers. Inaddition to the crude oils shown in Table XIII, various crude oils fromAlaska, Louisiana and Texas were emulsified by both Acinetobacterbioemulsifiers. Gas-oil was a better substrate for Acinetobacterbioemulsifier-induced emulsification than kerosene or gasoline, both ofwhich formed somewhat unstable emulsions. In general, better emulsionswere formed with α-emulsan than with β-emulsan and, in some instances,could only be formed with α-emulsan.

9.2. EMULSIFICATON OF PURE HYDROCARBONS

Straight and branch chain aliphatic hydrocarbons from heptane tooctadecane were emulsified only to a slight extent by the Acinetobacterbioemulsifier as illustrated by the data in FIG. 13 which is a graphicalrepresentation showing the relationship of the amount of emulsificationof various straight and branch chain alkanes as a function of carbonnumber. The data summarized in FIG. 13 was obtained using 100 mcg/ml ofAcinetobacter bioemulsifier and 0.05 ml hydrocarbon, the open circlesreferring to straight chain alkanes while the closed circles refer to2,2,5-trimethylhexane, 2-methyldecane, 2,6-dimethyldecane and2,6-dimethylunidecane. Increasing or decreasing the hydrocarbonconcentration by a factor of five did not improve emulsification.

Pentane and hexane were also not emulsified effectively; however,quantitative data for these two paraffins were not obtained because ofextensive evaporation during incubation. The solid hydrocarbons,nondecane, n-octacosane and hexatriacontane, were not dispersed byAcinetobacter bioemulsifier.

Emulsification of n-alkyl cyclohexane derivatives ranging frompropylcyclohexane to tridecylcyclohexane by Acinetobacter bioemulsifierare summarized in FIG. 14, which graphically illustrates emulsificationof various alkylcyclohexanes as a function of carbon number. The datashown in FIG. 14 was obtained using 0.2 ml hydrocarbon and either 25mcg/ml (closed circles) or 100 mcg/ml (open circles) of Acinetobacterbioemulsifier.

As shown in FIG. 14, two peaks of activity were observed, correspondingto octyl cyclohexane and decylcyclohexane. The data for octyl, nonyl anddecylcyclohexanes were obtained from redistilled materials whichcontained no ultraviolet-absorbing impurities. Concentrations of octyland decylcyclohexane as low as 5 mg per ml were rapidly and completelyemulsified by 50 mcg/ml of bioemulsifier. Nonylcyclohexane did notcontain any apparent inhibitors of emulsification, since mixtures ofoctyl and nonylcyclohexane were emulsified to about the same extent asoctylcyclohexane alone. Bicyclohexane and decalin were not emulsifiedsignificantly.

Emulsification of n-alkylbenzene derivatives by Acinetobacterbioemulsifier are summarized in FIG. 15, the data for which was obtainedusing 0.01 ml hydrocarbon and 50 mcg/ml of bioemulsifier. Maximumactivity was obtained with hexyl and heptylbenzenes. The total number ofcarbon atoms in the side chains may be more crucial than the chainlength since p-diisopropylbenzene behaved like hexylbenzene. The lowmolecular weight benzene derivatives, toluene, p-xylene, m-xylene,ethyl-benzene and 1,2,3,4-tetramethylbenzene, were not emulsifiedsignificantly. Aromatic compounds containing more than one ring,naphthalene, biphenyl, phenanthrene, anthracene, 3-phenyltoluene,1-methylnaphthalene and 2-methylnaphthalene were also not emulsifiedsignificantly by the Acinetobacter bioemulsifier.

9.3. EMULSIFICATION OF MIXTURES OF PURE HYDROCARBONS

Table VIII summarizes a number of experiments in which the Acinetobacterbioemulsifier-induced emulsification of aliphatic, aromatic and cyclichydrocarbons were measured in the presence of hexadecane or1-methylnaphthalene. Although neither the aliphatic compounds nor1-methylnaphthalene were emulsified by themselves, all mixturescontaining the aromatic compound and one of the aliphatic hydrocarbonswere excellent substrates for emulsification by the bioemulsifier. Theability of aromatic compounds to stimulate emulsification of aliphaticswas not limited to 1-methylnaphthalene, but occurred with toluene,p-xylene, 3-phenyltoluene and 2-methylnaphthalene. Addition ofhexadecane to the aliphatic compounds did not stimulate emulsification,that is, only an additive effect was observed. The minor exception tothis finding was non-adecane which became liquid when mixed withhexadecane.

As mentioned above, the only aromatic compounds that served assubstrates for emulsification by Acinetobacter bioemulsifier werealkylbenzene derivatives containing six or seven carbon atoms on theside chain(s). Aromatic compounds containing less than six carbon atomson the side chain were converted into good substrates for emulsificationby addition of hexadecane. Hexylbenzene and diisopropylbenzene wereconverted into even better substrates for emulsification by addition ofhexadecane. On the other hand, heptyl, decyl and pentadecylbenzene wereemulsified more poorly in the presence of hexadecane than by themselves.Only alkylbenzene derivatives containing side chains of five or morecarbon atoms were activated by 1-methylnaphthalene.1,2,3,4-tetramethylbenzene was poorly emulsified by the bioemulsifiereven in the presence of hexadecane or 1-methylnaphthalene. With fewexceptions, cycloparaffin derivatives were converted into bettersubstrates for Acinetobacter bioemulsifier-mediated emulsification byaddition of either hexadecane or 1-methylnaphthalene. In general,cyclohexane derivatives with short side chains (e.g., ethylcyclohexane)were activated more efficiently with aliphatic than aromatic compounds,while derivatives with long side chains (e.g., duodecylcyclohexane)formed better emulsions in the presence of 1-methylnaphthalene thanhexadecane. Dicyclohexane behaved like an aromatic compound in that itwas emulsified by the bioemulsifier in the presence of hexadecane butnot 1-methylnaphthalene. The fused dicylic compound decalin could not beemulsified by the bioemulsifier even by addition of hexadecane or1-methylnaphthalene.

Acinetobacter bioemulsifier-induced emulsion formation as a function ofthe relative concentrations of aliphatic (hexadecane) and aromatic(methylnaphthalene) compounds is shown in FIG. 16, the data for whichwas obtained using 50 mcg/ml of bioemulsifier and 0.05 ml of variousmixtures of hexadecane and 1-methylnaphthalene (closed circles) orhexadecane and 2-methylnaphthalene (open circles). Using either1-methylnaphthalene or 2-methylnaphthalene, maximum emulsion wasobtained with 25 vol. % hexadecane. Over fifty percent maximum emulsionwas obtained with ratios of hexadecane/metylnaphthalene from 4:1 to 1:6.An identical experiment using decane in place of hexadecane yieldedsimilar curves except that the peak of emulsion activity was obtainedwith 33 vol. % decane.

                  TABLE VIII                                                      ______________________________________                                        Emulsification of Mixtures of Aliphatic, Aromatic                             and Cyclic Hydrocarbons by Acinetobacter Bioemulsifier                                      Emulsion (Klett units)                                                                    plus    plus 1-                                                     no        hexa-   methylnaph-                                 Hydrocarbon.sup.a                                                                             addition  decane  thalene                                     ______________________________________                                        Aliphatics                                                                    decane          15            41    185                                       tetradecane     13            50    216                                       hexadecane      20            31    284                                       nonadecane      0     (solid) 79    285                                       2,2,5-trimethylhexane                                                                         0             34    89                                        2,6-dimethylunidecane                                                                         0              2    105                                       Aromatics                                                                     biphenyl        0     (solid) 123.sup.b                                                                           .sup. 19.sup.b                            naphthalene     0     (solid) .sup. 96.sup.b                                                                      .sup. 26.sup.b                            phenanthrene    0     (solid) .sup. 61.sup.b                                                                      .sup. 36.sup.b                            toluene         22            97     4                                        3-phenyltoluene 0             157    0                                        1-methylnaphthalene                                                                           0             284    0                                        2-methylnaphthalene                                                                           0             244    0                                        p-xylene        22            75    15                                        ethylbenzene    9             117   21                                        propylbenzene   9             90    23                                        pentylbenzene   4             197   85                                        hexylbenzene    98            188   165                                       p-diisopropylbenzene                                                                          96            299   192                                       heptylbenzene   105           82    186                                       decylbenzene    38            31    49                                        pentadecylbenzene                                                                             21              0    5                                        1,2,3,4-tetramethylbenzene                                                                    28            35     9                                        Cycloparaffins                                                                ethylcyclohexane                                                                              8             81    43                                        propylcyclohexane                                                                             3             81    64                                        butylcyclohexane                                                                              0             111   57                                        hexylcyclohexane                                                                              5              9    116                                       heptylcyclohexane                                                                             1             32    131                                       octylcyclohexane                                                                              109           151   175                                       nonylcyclohexane                                                                              0              0    249                                       decylcyclohexane                                                                              79            192   171                                       duodedecylcyclohexane                                                                         5              0    72                                        decalin         0             15    17                                        dicyclohexane   14            201   39                                        ______________________________________                                         .sup.a Experiments were performed using 50 mcg/ml of β-emulsan and       0.025 ml of each hydrocarbon (20 mg for solids).                              .sup.b For solubility reasons, 0.05 ml solutions containing 10% biphenyl,     10% naphthalene and 5% phenanthrene in hexadecane or 1methylnaphthalene       were used.                                                               

9.4. EFFECT OF ADDITION OF ALIPHATIC AND AROMATIC COMPOUNDS ONEMULSIFICATION OF PETROLEUM FRACTIONS

The results shown in Table VIII and summarized in FIG. 16 lead to theconclusion that the ability of the Acinetobacter bioemulsifiers toemulsify hydrocarbons depends on the relative concentrations ofaliphatic, cyclic and aromatic components in the hydrocarbon substrate.To verify this conclusion, experiments were designed to determinewhether or not addition of hexadecane or methylnaphthalene could enhanceAcinetobacter bioemulsifier-induced emulsification of petroleumfractions which had been fractionated to separate a fraction rich inaliphatics (Fraction 1) from two fractions (Fractions 2 and 3) rich inaromatics. These experiments, which are more fully described below inSection 13.14, show that the ability of α-emulsan to emulsify bothkerosene and gasoline was enhanced greatly by 2-methylnaphthalene butnot by hexadecane. Addition of even one part of the aromatic compound toten parts of gasoline or kerosene resulted in a much improved substratefor emulsification. The requirement for both aliphatic and aromaticconstituents was further supported by studying emulsification of columnfractionated crude oil. Although crude oil itself is emulsified by theAcinetobacter bioemulsifier, none of the fractions were good substratesby themselves. However, mixtures containing one fraction (Fraction 1)rich in aliphatics and the other (Fractions 2 or 3) rich in aromaticswere efficiently emulsified.

10. SUMMARY OF DIFFERENCES BETWEEN α-EMULSANS AND β-EMULSANS

The major differences between α-emulsans and β-emulsans, the two classesof bioemulsifiers produced by Acinetobacter Sp. ATCC 31012, may besubdivided into (a) differences in yield; (b) differences in structure;and (c) differences in emulsifying activity. Table IX summarizes severalof such differences between α-emulsans, β-emulsans and their respectivedeproteinized derivatives. The particular α-emulsans referred to inTable IX were prepared by growing Acinetobacter Sp ATCC 31012 on ethanolmedia, while the β-emulsans were prepared from an identical fermentationmedia using identical growth conditions except that hexadecene wassubstituted for ethanol. Both bioemulsifiers were purified by ammoniumsulfate fractionation, and the deproteinized derivative of eachbio-emulsifier was prepared by hot phenol extraction and furtherpurified prior to analysis. Total fatty acids content was determinedusing the hydroxamic acid test, taking the average equivalent weight ofthe fatty acid esters to be 230.

                  TABLE IX                                                        ______________________________________                                        Differences Between α-Emulsans and β-Emulsans                      and Their Respective Deproteinized Derivatives                                                   Specific                                                             Yield    Activity.sup.b    A/B                                      Bioemulsifier.sup.a                                                                     (mg/ml)  (units/mg)                                                                              % Esters.sup.b                                                                        Ratio.sup.c                              ______________________________________                                        'Emulsans 1-5      200-350   --      --                                       Apo-α-emulsans                                                                    --       100-200   8-14    0.2-0.5                                  β-Emulsans                                                                         0.1-0.75  50-200   --      --                                       Apo-β-emulsans                                                                     --       25-75     3-3     >0.8                                     ______________________________________                                         .sup.a αEmulsan was prepared from an ethanol medium and protoemulsa     from a hexadecane medium. Both bioemulsifiers were purified by ammonium       sulfate fractionation. The deproteinized derivatives of each bioemulsifie     were prepared by hot phenol extraction and further purified prior to          analysis.                                                                     .sup.b Total fatty ester content was determined using the hydroxamic acid     test, taking the average equivalent weight of the fatty acid esters to be     230.                                                                          .sup.c A and B refer to 2hydroxydodecanoic acid and 3hydroxydodecanoic        acid, respectively.                                                      

10.1. DIFFERENCES IN YIELD

As shown by Table IX and as further illustrated in the data summarizedin FIGS. 2 and 3, the yield of α-emulsan is invariably greater than theyield of β-emulsan even when identical cultures of Acinetobacter Sp.ATCC 31012 are used as innocula on ethanol and hexadecane media,respectively. Moreover, when the organism is grown on other carbonsources which produce α-emulsans, such as palmitic acid and dodecane,the yields of the high-ester α-emulsan are higher than the β-emulsansobtained when the organism is grown on such carbon sources aspentadecane or hexadecane.

10.2. DIFFERENCES IN STRUCTURE

Purified α-emulsans have a higher specific activity than purifiedβ-emulsans, which is probably due to the higher fatty acid ester contentof α-emulsans and may also be due to the generally higher amount of3-hydroxydodecanoic acid in α-emulsans compared to β-emulsans. As shownin Table IX, the apo-α-emulsan component of the α-emulsans containedfrom 8 to 14% by weight of total esters, while the apo-β-emulsancomponent of the β-emulsans contained appreciably less (2-3%) fatty acidesters. Moreover, the apo-α-emulsan content of α-emulsans generallypossess a lower ratio of 2-hydroxydodecanoic acid to 3-hydroxydodecanoicacid (usually about 1:4 to about 1:2) than in the apo-β-emulsancomponent of β-emulsans.

Table X summarizes the different ester compositions of an apo-α-emulsanderived from deproteinization of an α-emulsan formed when AcinetobacterSp. ATCC 31012 was grown on an ethanol medium when compared to theapo-β-emulsan derived from a β-emulsan formed when the organism wasgrown on hexadecane. Each of the deproteinized Acinetobacterbioemulsifiers was hydrolyzed in KOH/methanol for 4 days at roomtemperature, the corresponding mixture of methyl esters were formed withdiazomethane and the methyl esters of each mixture were thenfractionated by chromatography.

                  TABLE X                                                         ______________________________________                                        Ester Composition of Apo-α-emulsan and Apo-β-emulsan                              Apo-α-emulsan                                                                         Apo-β-emulsan                               Fatty Acid     (% Wgt)       (% Wgt)                                          ______________________________________                                        Decanoic       0.84          0.39                                             Dodecanoic     1.70          0.41                                             Dodecenoic     0.18          0.08                                             2-Hydroxydodecanoic                                                                          0.78          0.44                                             3-Hydroxydodecanoic                                                                          2.92          0.54                                             Hexadecenoic   0.05          trace                                            Hexadecenoic   trace         trace                                            Octadecenoic   0.02          trace                                            Octadecenoic   trace         trace                                            Unidentified   0.89          0.53                                             TOTAL ESTERS   7.4           2.4                                              ______________________________________                                    

The data shown in Table X confirm the general rule that theapo-α-emulsan content of α-emulsans, the aggregate amount of2-hydroxydodecanoic acid and 3-hydroxydedecanoic acid is usually about50% of the total fatty acid esters and may be as high as 70% of thefatty acid esters in the lipopolysaccharide.

10.3. DIFFERENCES IN EMULSIFYING ACTIVITY

The data contained in Table XIII below show that although α-emulsan andβ-emulsan are both excellent emulsifiers for crude oils and are bothonly fair emulsifiers for kerosenes, α-emulsan is much more effectivethan β-emulsan in the emulsification of gas-oils. Moreover, Bunker Cfuel oil is emulsified by α-emulsan but not by β-emulsan. In general,experience has shown that α-emulsans give better emulsions thanβ-emulsans with hydrocarbon substrates which contain both aliphatic andaromatic (or cyclic) components.

11. SORPTIVE PROPERTIES OF EMULSANS AND THEIR DERIVATIVES ON SOLIDSUBSTRATES

The adsorption or non-adsorption of emulsans and apoemulsans on varioustypes of solid substrates, such as sand, limestone or clay minerals,were measured to determine whether these anionic lipopolysaccharidescould function as bioemulsifiers in the presence of such solidsubstrates.

11.1. NON-ADSORPTION ON SAND AND LIMESTONE

Neither emulsans nor apoemulsans are adsorbed to any significant extenton sand or on limestone over the pH range in which these bioemulsifierswill be used to form oil-in-water emulsions. When oil is present on thesand or limestone, such as in sand or sandstone reservoir formations orin limestone reservoir formations, the oil may be recovered by enhancedoil recovery using chemical flooding with dilute concentrations ofemulsan, since bench scale experiments have shown that whenoil-saturated sand or oil-saturated limestone are treated with dilutesolutions (i.e., from 0.1 to 0.5 mg/ml) of α-emulsan containingmagnesium ions (10 mM), over 90% of the oil can be removed from theoil-saturated sand and over 98% of the oil can be removed from theoil-saturated limestone. Comparable results may be obtained using seawater solutions of emulsans, since the presence of sodium chloride inthe concentrations found in sea water or in connate water do not affectthe ability of emulsans to emulsify crude oils, including crude oilswhich are quite viscous or tarry, which are found in sand (or sandstone)formations or in limestone formations or which remain in such formationsafter secondary recovery techniques (such as steam stripping) areemployed.

11.2. ADSORPTION ON ALUMINOSILICATE CLAYS

Emulsans and their deproteinized derivatives, the apoemulsans, both ofwhich are strongly anionic, are adsorbed on aluminosilicateion-exchangers, such as kaolin, bentonite and other clay minerals whichhave ion-exchange capacity.

The kinetics of adsorption of α-emulsan on bentonite are shown in FIG.17, which summarizes the rate of adsorption of α-emulsan onto 0.5 gbentonite in a 20 ml solution of 20 mM Tris-Mg buffer [20 mMtris(hydroxymethyl)aminomethane hydrochloride and 10 mM magnesiumsulfate] containing 100 mcg/ml of α-emulsan. The mixture was shaken at20° C. at 110 strokes per minute in 100 ml flasks, with samples beingremoved every 10 minutes for assay of α-emulsan not bound to thebentonite. Under these conditions, over 95% of the α-emulsan wasadsorbed and equilibrium reached in 40 minutes. The amount of α-emulsanadsorbed by the aluminosilicate clay was a function of the amount ofclay, about 70% of the α-emulsan being adsorbed at a bentonite/emulsanratio of 100:1 and over 95% of the α-emulsan at ratios over 400:1.

11.3. FLOCCULATION OF CLAYS

Adsorption of emulsans (as well as the apoemulsans, ψ-emulsans andproemulsans) onto suspended particles of aluminosilicate clays, such askaolin and bentonite, results in rapid flocculation of such particles.By way of illustration, mixing 1 g of bentonite with 20 ml watercontaining only 100 mcg/ml of α-emulsan causes the bentonite to sedimentfrom five to ten times faster than in the absense of emulsan. Moreover,the supernatant fluid obtained using emulsan-mediated flocculation wasclear, while the sedimentation of bentonite without the emulsan yieldedan upper layer which remained opalescent even after prolonged standing.Similar results may be obtained with other clay minerals withion-exchange capacity.

The flocculating properties of emulsans (which apply equally to thecorresponding apoemulsans, as well as to their deacylated derivatives,ψ-emulsans and proemulsans, all of which are also anionic) prevent thepacking of aluminosilicate clays into a dense precipitate in such mannerthat the volume occupied by the flocculated clays is several timesgreater (three times in the case of bentonite) than in the absense ofthe lipopolysaccharide. The flocculated aluminosilicate clays now havecertain fluid and flow properties which suggest an enormous number ofuses for emulsans and apoemulsans and their derivatives in flocculation,including (a) the use of emulsans and apoemulsans as a clay particleflocculent in drilling muds; (b) the prevention of clogging in sewagetreatment systems; (c) enhancing the porosity of clay solids tostructure poor soils for uses in agriculture; (d) the inclusion ofemulsans in coatings and aerosol sprays containing such clays; and (e)the use of emulsans and apoemulsans as a general flocculating agent forrecovery and settling processes.

11.4. RELATIONSHIP OF FLOCCULATION TO BREAKING OIL/WATER EMULSIONS

Adsorption of emulsans onto aluminosilicate clays creates an oleophilicclay which, in turn, is capable of breaking a stable oil/water emulsionformed with the bioemulsifier. By way of illustration, emulsification of1 ml Agha Jari crude oil in 10 ml sea water containing about 0.1 mg/mlof α-emulsan forms an oil-in-water emulsion which is stable afterstanding two days. The addition of 1 g of preswelled bentonite to thisstable emulsion, followed by intense shaking for about 20 seconds,resulted in breakage of the emulsion in 15 minutes. After 20 hours,there were two separated layers, namely an upper clear liquid and alower gel-like sediment which occupied about one-half of the priorvolume of the emulsion.

These sorptive properties of emulsans and apoemulsans with respect toaluminosilicate clay ion-exchangers may also be utilized to remove oiland hydrocarbonaceous sludge from oily ballast water or other oilywater, either by filtering such oily waters through an aluminosilicateclay (such as kaolin or bentonite) on which an emulsan or apoemulsan hasbeen adsorbed or, alternatively, by adding the emulsan or apoemulsan tothe oily water and then filtering the mixture through an aluminosilicateclay. In both cases, the filtrate will be clear and the oily residuewill remain in the clay filter.

12. ENVIRONMENTAL AND ENERGY-RELATED USES

The emulsifying agents of the invention, which comprise an aqueoussolution in sea water or fresh water containing (1) from about 10 mcg/mlto about 20 mg/ml of α-emulsans, and (2) from about 1 to about 100 mM ofat least one divalent cation, such as magnesium, calcium or manganese,possess the combination of characteristics that permit these emulsifyingagents to be widely employed for several important environmental andenergy-related uses, namely cleaning oil-contaminated vessels, oil spillmanagement, and enhanced oil recovery by chemical flooding.

By way of illustration, hydrocarbonaceous residues (including residualpetroleum) may be cleaned from tankers, barges, storage tanks, tank carsand trucks, pipelines, and other containers used to transport or tostore crude oil or petroleum fractions, by washing the oil-contaminatedsurfaces of such vessels with the emulsifying agent, using an amount ofα-emulsan in the solution which can be predetermined based on thecomposition of the particular hydrocarbon to be removed. As a generalrule, complete cleaning can be accomplished with hydrocarbon/emulsanweight ratios of about 1000:1 to 10000:1, the higher the ratio the lessstable the emulsion. Moreover, the resultant oil-in-water emulsions canbe broken by physical or chemical techniques, and the oil recovered forfuel values or for refining.

Oil spill management is another environmentally important use for theemulsifying agents of the invention. In most processes for cleaning oilspills, an aqueous solution of a detergent or surfactant is brought intocontact with the oil slick, which is floating on the sea or which hasbeen washed ashore or deposited on land to emulsify the oil so that itmay be dispersed and either removed or biodegraded. Most of thedetergents or surfactants commonly used are somewhat toxic to marinelife and are not biodegradable. By using the emulsifying agents of theinvention, namely the aqueous solution in sea water or fresh watercontaining from about 10 mcg/ml to about 20 mg/ml of α-emulsans and aneffective concentration of at least one divalent cation, not only is itpossible to emulsify the oil with less emulsifier which is itselfbiodegradable but also to avoid toxological problems since emulsans arenon-toxic in the concentrations in which they are used asbioemulsifiers. This technique is especially useful in cleaning beachescontaminated with oil.

Enhanced oil recovery by chemical flooding represents a particularlyimportant energy-related use for α-emulsans. All processes in theenhanced recovery of oil by chemical flooding involve the injection of achemically-augmented "slug" comprising water and one or more addedchemicals into a petroleum reservoir followed by displacement of the"slug" through the reservoir to recover crude oil from the injectedreservoir. Because of the unique combination of properties of emulsansand particularly for α-emulsans--namely (a) that emulsans on aweight-for-weight basis are probably the most efficient oil-in-wateremulsifiers discovered; (b) that emulsans exhibit a high degree ofspecificity in emulsifying hydrocarbon substrates that contain aliphaticand aromatic or cyclic fractions, which are present in all crude oilsincluding the viscous and tarry crudes remaining in the reservoir afterprimary and secondary recovery; (c) that emulsans function effectivelyeven in the presence of high concentrations of salts, such as brine; and(d) that emulsans are not adsorbed to any significant extent by sand orsandstone or limestone--using a chemically-augmented slug which containseffective concentrations of α-emulsans and the necessary divalent cationwill appreciably increase the recovery of oil from sand or sandstone orlimestone formations. Moreover, these anionic lipopolysaccharides may beused as the sole emulsifier or in conjunction with other emulsifyingagents (such as the nonionic surfactants used for tertiary oilrecovery), as well as in conjunction with the mobility control polymersused in such processes.

13. EXAMPLES

The following examples are illustrative of the preparation, purificationand some of the uses of the α-emulsans and apo-α-emulsans derived fromAcinetobacter Sp. ATCC 31012 when compared to the β-emulsans andapo-β-emulsans which, in turn, are derived from growing the sameorganism on a different substrate. Except when otherwise indicated, theα-emulsans described in such examples were obtained by growing theorganism on an ethanol medium. Where used, the β-emulsans were obtainedby growing the organism on a hexadecane medium.

13.1. PREPARATION OF α-EMULSAN FROM ETHANOL IN FRESH WATER MEDIA

To a 60-liter fermenter fitted with four baffles and a variable-speedagitator were added 733.6 g of dibasic potassium phosphate [K₂ HPO₄.3H₂O], 240 g of monobasic potassium phosphate, 8 g of magnesium sulfate[MgSO₄.7H₂ O], 160 g of ammonium sulfate and a sufficient amount ofdeionized water to make 40 liters. The medium was sterilized for 40minutes at 121° C., after which 800 ml of absolute ethanol (2% byvolume) was added. The final pH of the medium was 6.9.

Growth was initiated with 2 liters (5%) of a late exponential culture ofAcinetobacter Sp. ATCC 31012 grown under similar fermentationconditions. The fermentation was conducted at 30° C., with aerationmaintained at 15 liters of air per minute and agitation at 250 rpm. ThepH of the fermentation broth was maintained between pH 6.2 and 6.7 bythe dropwise addition of concentrated ammonium hydroxide, which requiredapproximately 185 ml of concentrated ammonium hydroxide during the first30 hours.

Throughout the fermentation, foam was controlled by automatic pulseadditions of a silicone defoamer (Dow Corning 525, sterlizable, diluted1:8), in connection with which an aggregate of 50 ml was added duringthe first 30 hours. Commencing at the 11th hour of fermentation, ethanolwas continuously added to the fermentation broth at the rate of 40 mlper hour. Ammonium sulfate was periodically added to the fermentationbroth at the rate of 2 g per hour for the first 30 hours.

Maximum growth was obtained between 24 to 30 hours after inoculation.The yield of α-emulsan was 4 g per liter, with a cell mass ofapproximately 8 g (dry weight basis) per liter. Analysis of the crudeα-emulsan, which was performed on the crude extracellular fluidfollowing extensive dialysis against water, showed that it contained atotal ester content of 10% using the hydroxamic acid test and assumingthat the average molecular weight of the fatty acid esters was 230.Using substantially identical conditions, as much as 5.3 g per liter ofα-emulsan were obtained with a cell mass of about 9 g (dry weight basis)per liter.

13.2. PREPARATION OF α-EMULSAN FROM ETHANOL IN SEA WATER

Acinetobacter Sp. ATCC 31012 was grown in a 250 ml flask containing 40ml filtered sea water, 0.73 g dibasic potassium phosphate [K₂ HPO₄.3H₂O], 0.24 g monobasic potassium phosphate, 0.8 g urea, and 0.8 mlabsolute ethanol (2% by volume). The medium was inoculated with 2 ml ofa late exponential culture of Acinetobacter Sp. ATCC 31012 grown undersimilar conditions. Incubation was for 96 hours at 30° C., with gyratoryshaking at 250 rpm. After removal of the cells by centrifugation at10,000×g for 15 minutes and dialysis against water, analysis showed thatthe yield of crude α-emulsan was 120 units per ml with a specificactivity of 270 units per mg. The crude α-emulsan contained 13% totalester content when measured by the hydroxamic acid test, assuming theaverage molecular weight of the fatty acid esters to be 230.

13.3 PREPARATION OF α-EMULSAN FROM SODIUM PALMITATE

Acinetobacter Sp. ATCC 31012 was grown in an aqueous medium containing18.34 mg/ml of dibasic potassium phosphate [K₂ HPO₄.3H₂ O], 6 mg/ml ofmonobasic potassium phosphate, 0.2 mg/ml of magnesium sulfate [MgSO₄.7H₂O], 4 mg/ml of ammonium sulfate and 1.2 mg/ml of sodium palmitate.Growth was initiated by inoculating 0.1 ml of a washed cell suspensioninto 40 ml of the medium in a 250 ml flask. Incubation was for 72 hoursat 30° C., with gyrotary shaking at 250 rpm. After removal of the cellsand extensive dialysis of the crude extracellular fluid against water,analysis showed that the yield of the α-emulsan was 111 units per mlwith a specific activity of 116 units per mg determined by the standardassay technique. The crude α-emulsan contained 9% total ester contentwhen measured by the hydroxamic acid test, assuming the averagemolecular weight of the fatty acid esters to be 230.

13.4. PREPARATION OF α-EMULSAN FROM DODECANE

Acinetobacter Sp. ATCC 31012 was grown in an aqueous medium containing18.34 mg/ml of dibasic potassium phosphate [K₂ HPO₄.3H₂ O], 6 mg/ml ofmonobasic potassium phosphate, 0.2 mg/ml of magnesium sulfate [MgSO₄.7H₂O], 4 mg/ml of ammonium sulfate and 0.8 mg/ml of dodecane. Growth wasinitiated by inoculating 0.1 ml of a washed cell suspension into 40 mlof the medium in a 250 ml flask. Incubation was for 72 hours at 30° C.,with gyrotary shaking at 250 rpm. After removal of the cells andextensive dialysis of the crude extracellular fluid against water,analysis showed that the yield of the emulsan was 76 units per ml with aspecific activity of 81 units per mg determined by the standard assaytechnique. The crude emulsan contained 9% total ester content whenmeasured by the hydroxamic acid test, assuming the average molecularweight of the fatty acid esters to be 230.

13.5 PREPARATION OF β-EMULSAN FROM HEXADECANE

Using the medium described above in Section 13.4 with 0.2 mg/ml ofhexadecane being substituted as the primary assimilable carbon source inplace of dodecane, Acinetobacter Sp. ATCC 31012 was grown at 30° C. for72 hours, with gyrotary shaking at 250 rpm. As before, growth wasinitiated by inoculating 0.1 ml of a washed cell suspension into 40 mlof the medium in a 250 ml flask.

After removal of the cells and extensive dialysis of the crudeextracellular fluid against water, analysis showed that the yield of theβ-emulsan was 16 units per ml with a specific activity of 50 units permg determined by the standard assay technique. The crude protoemulsancontained almost 5% total ester content when measured by the hydroxamicacid test, assuming the average molecular weight of the fatty acidesters to be 230. The corresponding apo-β-emulsan, obtained by hotphenol extraction in accordance with the deproteinization techniquedescribed below in Section 13.7, contained an ester content between 2 to3% when measured by the hydroxamic acid test.

13.6 PREPARATION OF APO-α-EMULSAN

Various samples of emulsan contain between 5% to 15% protein by weight,which reflects the degree of purity of the bioemulsifier. In order toascertain whether or not the protein moeity was essential foremulsifying activity, α-emulsan which had been prepared by growingAcinetobacter Sp. ATCC 31012 on an ethanol medium was deproteinized bythe hot phenol method described by O. Westphal et al. in the monographedited by R. L. Whistler, "Carbohydrate Chemistry", Academic Press,Inc., New York, 1965, pp. 83-91.

One gram of such α-emulsan, dissolved in 200 ml water with the aid of afew drops of concentrated ammonium hydroxide, was brought to 65°-68° C.and then added to an equal volume of 90% phenol which had been preheatedto 65° C. The mixture was stirred vigorously for 15 minutes at 65° C.and then cooled to 10° C. in an ice bath. The resulting emulsion wascentrifuged at 5,000×g for 30 minutes. After transferring the viscousaqueous phase to a flask, the remaining phenol layer and interface wereextracted three more times with 200 ml water. The combined waterextracts were dialyzed extensively against several changes of distilledwater and then freeze-dried to obtain 850 mg (85% yield) ofapo-α-emulsan as a white fluffy solid.

The remaining phenol fraction and interphase were suspended in water,dialyzed extensively against distilled water and freeze-dried, yielding100 mg (10% yield) of a yellowish proteinaceous material whichrepresents the denatured protein derived from such α-emulsan.

The ability of each of these fractions to emulsify gas-oil was thendetermined using the standard assay technique. Emulsion formation wasmeasured in 125 ml flasks containing 7.5 ml Tris-Mg buffer [200 mMtris-(hydroxymethyl)aminomethane hydrochloride, pH 7.4; 10 mM magnesiumsulfate] 0.05 ml Gach-Saran gas-oil and either 75 mcg of α-emulsan, 75mcg of apo-α-emulsan or 15 mcg of the denatured protein obtained byphenol extraction of such emulsan. Flasks were agitated by reciprocalshaking (150 strokes per minute) for one hour at 26° C. Contents of theflasks were then transferred to Klett tubes for measurement of turbidityin a Klett-Summerson colorimeter fitted with a green filter. The resultsof these tests are summarized in Table XI, the specific activity(reported in units per mg dry weight) having been determined from thestandard curve (Curve 1-B) shown in FIG. 1.

                  TABLE XI                                                        ______________________________________                                        Emulsification of Gas-Oil                                                                               Specific Activity                                   Fraction      Amount (mcg)                                                                              (units per mg)                                      ______________________________________                                        α-Emulsan                                                                             75          276                                                 Denatured protein                                                                           15           0                                                  Apo-α-emulsan                                                                         75          146                                                 ______________________________________                                    

The data contained in Table XI show that all of the emulsifying activityis in the 0-lipoacyl heteropolysaccharide and that none of the activityis associated with the denatured protein fraction.

From additional experimental work on apo-α-emulsan, it was found thataddition of 0.2 and 2.0 mcg/ml of this denatured protein to 10 mcg/ml ofapo-α-emulsan resulted in 25% and 66% "stimulations" of emulsifyingactivity, respectively, which actually is a measure in the amount ofturbidity obtained in the standard emulsifier assay which, in turn, isbelieved to be related to emulsifying activity. This increase inturbidity of hydrocarbon substrate when protein was added toapo-α-emulsan was not specific to the denatured protein derived byphenol extraction of α-emulsan, since different proteins, such as bovineserum albumin, lysozyme, hexokinase and denatured alcohol dehydrogenase,also result in increased turbidities in the emulsification of gas-oilwhen such proteins are added to apo-ψ-emulsan.

13.7. PREPARATION OF APO-β-EMULSAN

The hot phenol method of O. Westphal et al., supra, may also be used toextract the associated protein contained in β-emulsan and thereby formthe corresponding apo-β-emulsan. Using the experimental method describedabove in Section 13.5, the β-emulsan which had been prepared by growingAcinetobacter Sp. ATCC 31012 on a hexadecane medium was deproteinized toform the corresponding apo-β-emulsan. All of the emulsifying activitywas found to be in the 0-lipoacyl heteropolysaccharide and none of suchactivity was found to be associated with the denatured protein fraction.

13.8. PREPARATION OF ψ-EMULSAN

Mild base hydrolysis of emulsans will 0-deacylate the lipopolysaccharidewithout affecting the N-acyl groups, which technique may be used toprepare the ψ-emulsans. Ten milliliters of an aqueous solutioncontaining 2.5 mg/ml of α-emulsan were treated with an equal volume of0.2 M NaOH at 98° C. for 2 hours. The solution was then cooled in an icebath and carefully neutralized to pH 7.0. The neutralized solution wasextensively dialyzed against water and lyophilized, yielding 20 mg (80%)of ψ-emulsan having a Specific Emulsifying Activity of 76 units per mg.The total ester content of the ψ-emulsan was 1% by the hydroxamic acidtest. The reduced viscosity of this ψ-emulsan was 317 cc/gram.

13.9. PREPARATION OF PROEMULSAN

Base hydrolysis of the emulsans, apoemulsans or ψ-emulsan willcompletely 0-deacylate and partially N-deacylate the biopolymer,hydrolyzing any associated protein at the same time. The resultantproducts are the proemulsans. Fifty mg of apo-α-emulsan in 30 ml of 2%KOH in methanol solution were left at room temperature for 96 hours.After removal of the methanol at low ressure, 15 ml of water were addedand the pH adjusted to ph 2.0. the free acids were removed by etherextraction, and the aqueous solution was dialyzed and lyophilized,yielding 37 mg (74%) of proemulsan. The ester content of the proemulsan,as assayed by the hydroxamic acid test, was zero. Moreover, the producthas no emulsification activity when assayed by the standardemulsification test. Elemental analysis: C 36.5%, H 7.0%, N 6.5%.

13.10. PURIFICATION OF α-EMULSAN BY PRECIPITATION WITH AMMONIUM SULFATE

A late exponential culture (1:1000 dilution) of Acinetobacter Sp. ATCC31012 was grown at 30° C. in a New Brunswick 14-liter fermenter using anaqueous medium containing 14 g per liter of dibasic acid potassiumphosphate [K₂ HPO₄.3H₂ O], 6 g per liter of monobasic potassiumphosphate, 0.2 g per liter of magnesium sulfate [MgSO₄.7H₂ O], 4 g perliter of ammonium sulfate and 20 ml per liter of absolute ethanol. Thefermentation was conducted using aeration at about 15 liters per minuteand agitation at 100 rpm without baffles, adding ethanol as required.

When the fermentation had proceeded about 3 days, the medium was allowedto cool and 1760 g of ammonium sulfate were added slowly, with stirring,directly to 10-liters of cooled fermentation broth without prior removalof the cells (30% ammonium sulfate saturation). After standingovernight, the supernatant fluid was collected by decantation. Theprecipitate was suspended in 30% saturated ammonium sulfate andcentrifuged at 10,000×g for 15 minutes. The combined supernatant fluidswere further clarified by passage through a thin layer of Kieselgel. Tothe cell-free supernatant fluid was added an additional portion (62 gper liter) of ammonium sulfate to reach a final concentration of 40%saturation.

The resulting precipitate, collected by centrifugation at 10,000×g for15 minutes, was dissolved in 200 ml of water, extracted with ether,dialyzed against distilled water and lypophilized. The yield ofα-emulsan was 2.1 g from 10-liters of fermentation broth, with aSpecific Emulsification Activity of 330 units per mg.

13.11. PURIFICATION OF α-EMULSAN BY PRECIPITATION WITH QUATERNARYAMMONIUM SALTS

One gram of crude α-emulsan was dissolved in 100 ml of water to yield aclear viscous solution. Twenty milliliters of a 5% w/v aqueous solutionof cetyltrimethyl ammonium bromide was added with mixing at roomtemperature. After allowing the precipitate to aggregate a few minutes,the mixture was centrifuged at 5,000×g for 10 minutes. The pelletfraction, which contained all the emulsifying activity, was washed oncewith distilled water. The washed cetyltrimethyl ammonium bromideprecipitate was dissolved in 100 ml of 0.1 M sodium sulfate. A smallamount of precipitate remaining was removed by centrifugation at10,000×g for 30 minutes. One gram of potassium iodide was then added tothe clear solution with mixing. The cetyltrimethyl ammonium iodideprecipitate that formed was removed by centrifugation at 10,000×g for 15minutes. The remaining supernatant fluid was dialyzed extensivelyagainst distilled water and lyophilized to yield a white solid. Thismaterial had a Specific Emulsification Acitvity of 350 units per mg.

A sample of the CTAB-purified α-emulsan was subjected to said hydrolysisat 98° C. in 5 M HCl for 6 hours to liberate any glucose that may havebeen present in the biopolymer. The hydrolyzed material was thenanalyzed by thin layer chromatography on a cellulose-F plate; silvernitrate staining showed only a trace of glucose, probably as animpurity.

13.12 PURIFICATION OF β-EMULSAN BY HEPTANE PARTITIONING

Using the medium described above in Section 13.10 with 0.2% (v/v)hexadecane being substituted as the primary assimilable carbon source inplace of ethanol, Acinetobacter Sp. ATCC 31012 was grown at 30° C. inNew Brunswick 14-liter fermenters for 4 days.

Twenty-seven liters of the hexadecane-grown culture were cooled and thecells removed by centrifugation in a Sorvall KSB continuous flowcentrifuge. The supernatant fluid was then extracted twice with 1/3volume of ether. Residual ether in the aqueous phase was removed bybubbling with filtered nitrogen gas. The ether phase contained nomeasurable emulsifying activity and was discarded.

The aqueous phase was filtered successively through 3, 1.2, 0.8 and 0.45micron Millipore filters, and the clear filtrate was then extracted fourtimes with 0.15 volume heptane. Approximately 10% of the emulsifyingactivity which remained in the aqueous phase was discarded.

The heptane fractions were combined and evaporated to a yellow syrup invacuo. After extraction with ether, the syrup was dissolved in 100 ml of50% aqueous methanol. The resulting viscous solution was dialyzedagainst several changes of distilled water and lyophilized. The yield oflyophilized β-emulsan was 1.5 g, with an extraordinarily high specificactivity of 205 units per mg.

A sample of this material was subjected to base hydrolysis for 72 hoursat room temperature, using an aqueous solution of 90% methanolcontaining 2.5% KOH. After removal of the methanol in vacuo, addition ofwater and acidification to pH 1, the fatty acids were extracted withether, methylated with diazomethane and were then subjected to gaschromatographic analysis. The chromatograph revealed the presence of2-hydroxydodecanoic acid (A) and 3-hydroxydodecanoic (B) acid in aweight ratio of A/B equal to 0.83.

13.13. AMMONIUM SULFATE FRACTIONATION OF APO-α-EMULSAN

The phenol extraction method described above in Section 13.6 wasrepeated on 820 mg of α-emulsan. After three phenol extractions, thecombined water extracts were extracted four times with an equal volumeof ether to remove residual phenol. Following evaporation of ether, theviscous aqueous phase was cooled to 5° C. and brought to 32.5% ammoniumsulfate saturation, no precipitation having formed at 30% saturation.After standing for one hour at 5° C., the clear translucent precipitatewas collected by centrifugation at 5,000×g for 30 minutes at 5° C.

The procedure was repeated to obtain a slightly turbid secondprecipitate between 32.5% and 35% saturation and another smallprecipitate between 35% and 40% saturation. No additional precipitateformed between 40% and 60% saturation. Each of the precipitates wasdissolved and was dialyzed at 2°-5° C. successively against distilledwater, 0.05 N hydrochloric acid (24 hours) and double distilled water.The same procedure was also followed with the remaining 60% saturatedsolution. Each of the resulting solutions remaining after suchpurification was freeze-dried and analyzed. The results of such analysesare set forth in Table XII.

The analytical data contained in Table XII show that over 99% of theemulsifying activity of apo-α-emulsan precipitated in the two fractionsbetween 30% and 35% ammonium sulfate saturation. These two apo-α-emulsanfractions were characterized by similar Specific EmulsificationActivities and had the same proportions of 0-ester, carboxylic acid andhexose. Moreover, both of the active fractions had high specificviscosities. None of the fractions contained significant quantities ofprotein.

                                      TABLE XII                                   __________________________________________________________________________    Analyses of Ammonium Sulfate-Precipitated Fractions of Apo-α-emulsan    Ammonium                                                                      Sulfate                                                                       Concen-                                                                       tration (%)                                                                   at which    Emulsifying            Carboxylic                                 Precipi-                                                                            Weight of                                                                           Activity Reduced  O--Ester                                                                           Acid  Hexose.sup.a                         tation                                                                              Precipi-                                                                            Klett                                                                             Specific                                                                           Viscosity                                                                          Protein                                                                           (μ moles                                                                        (μ moles                                                                         (μ moles                          Occurred                                                                            tate (mg)                                                                           Units                                                                             Activity                                                                           (cc/g)                                                                             (%) per mg)                                                                            per mg)                                                                             per mg)                              __________________________________________________________________________      30-32.5                                                                           379   66,500                                                                            175  810  0.3 0.66 1.5   0.27                                 32.5-35                                                                             194   34,500                                                                            178  570  0.15                                                                              0.63 1.5   0.33                                 35-40  25   780  31  400  0.5 0.81 --    0.20                                 40-60  82   0    0   --   0.7 --   --    0.08                                 __________________________________________________________________________     .sup.a The small amounts of hexose (glucose equivalents) which were           detected are due to the presence of a small amount of a contaminating         material which coprecipitated with the apoα-emulsan, but which coul     be removed following fractionation of the apo -emulsan with cetyltrimethy     ammonium bromide. This contaminating material was a lipopolysaccharide        which contained glucose. It had no emulsifying activity when assayed by       the standard emulsification technique.                                   

13.14. EMULSIFICATION OF PETROLEUM FRACTIONS BY α-EMULSANS ANDβ-EMULSANS

The presence of a higher 0-lipoester content in α-emulsans compared toβ-emulsans results in significant differences in the emulsificationactivity of these Acinetobacter bioemulsifiers. This conclusion wasdemonstrated by a series of test which were conducted to determine theeffect of both dioemulsifiers on various types of petroleum fractionswhich are widely used within and sold by the oil industry.

In each of these tests, emulsion formation was measured in 125 mlrubber-stoppered flasks containing 5 ml of filtered sea water, 8 mg/mlof hydrocarbon and 50 mcg/ml of the particular Acinetobacterbioemulsifier, the α-emulsan having been prepared by growingAcinetobacter Sp. ATCC 31012 on an ethanol medium while the β-emulsanwas prepared by growing the organism on a hexadecane medium. Theα-emulsans were purified by the ammonium sulfate fractionation techniquedescribed above in Section 13.10 while the β-emulsans were purified bythe heptane partitioning technique described above in Section 13.12.

Flasks were agitated by gyratory shaking (280 rpm) or by reciprocalshaking (150 strokes per minute) for 2 hours at 25° C. Contents of theflask were then transferred to Klett tubes for measurement of turbidityin a Klett-Summerson colorimeter fitted with a green filter. Readingswere taken after standing undisturbed for 10 minutes. Controls lackingeither the particular Acinetobacter emulsifier or hydrocarbon yieldedreadings of less than 5 Klett units. The results of these tests aresummarized in Table XIII.

                  TABLE XIII                                                      ______________________________________                                        Emulsification of Petroleum Fractions by                                      α-Emulsans and β-Emulsans                                          Petroleum Fraction                                                                         Emulsifier Emulsion  (K.U.)                                      (8 mg/ml)    (50 mcg/ml)                                                                              Gyratory  Reciprocal                                  ______________________________________                                        Crude Oils                                                                    Darius       α-Emulsan                                                                          650       1090                                        Agha Jari    α-Emulsan                                                                          720       950                                         Agha Jari    β-Emulsan                                                                           780       --                                          Rostam       β-Emulsan                                                                           758       --                                          Gas-Oils                                                                      Darius       α-Emulsan                                                                          300       800                                         Gach Saran   α-Emulsan                                                                          --        500                                         Belayim Marine                                                                             α-Emulsan                                                                          100       --                                          Agha Jari    α-Emulsan                                                                          195       840                                         Agha Jari    β-Emulsan                                                                           --        420                                         Kerosenes                                                                     Darius       α-Emulsan                                                                           42       160                                         Belayim Marine                                                                             α-Emulsan                                                                           35       --                                          Agha Jari    α-Emulsan                                                                           41       110                                         Agha Jari    β-Emulsan                                                                           --        125                                         Miscellaneous                                                                 Diesel Oil   α-Emulsan                                                                          290       --                                          Diesel Oil   β-Emulsan                                                                           --        490                                         Bunker C Fuel Oil                                                                          α-Emulsan                                                                          --        680                                         Bunker C Fuel Oil                                                                          β-Emulsan                                                                           --         35                                         Light Petroleum Oil                                                                        β-Emulsan                                                                           --        218                                         Gasoline (83 Octane)                                                                       α-Emulsan                                                                          --         89                                         ______________________________________                                    

Analysis of the data contained in Table XIII show that althoughα-emulsan and β-emulsan are both excellent emulsifiers for crude oilsand are both only fair emulsifiers for kerosenes, α-emulsan is much moreeffective than β-emulsan in the emulsification of gas-oils. In fact,emulsions of gas-oils were as stable as crude oil emulsions, the majorreason for the higher Klett readings of crude oil emulsions than thosefor gas-oil emulsions being the dark color of crude oil compared togas-oil. Bunker C fuel oil was emulsified by α-emulsan but not byβ-emulsan. Considering that the darker color of crude oil may haveobscured the relative emulsification activities of both bioemulsifiers,the data show that in general better emulsions were obtained withα-emulsan than with β-emulsan and with reciprocal rather than withgyratory shaking.

13.15. EMULSIFICATION OF MIXTURES OF PETROLEUM FRACTIONS AND PUREHYDROCARBONS BY α-EMULSAN

To determine whether emulsans exhibit any specificity in theemulsification of different types of hydrocarbons, a series of testswere conducted to measure the effect of α-emulsan in the emulsificationof mixtures of various petroleum fractions and pure hydrocarbons.

In each of these tests, emulsion formation was measured in 125 mlrubber-stoppered flasks containing 5 ml of filtered sea water, 8 mg/mlof total substrate (petroleum fraction plus additive) and 50 mcg ofα-emulsan. All mixtures of hydrocarbons were 1:1 (v/v). In some of thetests, fractions of Agha Jari crude oil were used, the fractions havingbeen prepared by the procedure of A. Jobson et al., App. Microbiol., 23,1082-1089 (1972), under which procedure Fractions 1, 2 and 3 correspondto the aliphatic (saturates), aromatic and polar aromatic fractions,respectively. As before, the α-emulsan was prepared by growingAcinetobacter Sp. ATCC 31012 on an ethanol medium and was purified bythe ammonium sulfate fractionation technique.

Flasks were agitated by reciprocal shaking (150 strokes per minute) for2 hours at 25° C. Contents of the flask were then transferred to Kletttubes for measurement of turbidity in a Klett-Summerson colorimeterfitted with a green filter. Readings were taken after standingundisturbed for 10 minutes. The results of these tests are summarized inTable XIV.

                  TABLE XIV                                                       ______________________________________                                        Emulsification of Mixtures of Petroleum                                       Fractions and Pure Hydrocarbons by α-Emulsan                            Petroleum Fraction                                                                         Additive       Emulsion (K.U.)                                   ______________________________________                                        Kerosene     none           190                                               Kerosene     hexadecane      68                                               Kerosene     2-methylnaphthalene                                                                          1050                                              Gasoline     none           115                                               Gasoline     hexadecane     230                                               Gasoline     2-methylnaphthalene                                                                          1100                                              Agha Jari                                                                     Fraction 1   none           130                                               Fraction 2   none            60                                               Fraction 3   none           105                                               Fraction 1   Fraction 3     1050                                              Fraction 2   Fraction 3     1500                                              Fraction 3   Fraction 3      80                                               ______________________________________                                    

The data contained in Table XIV show that the efficacy of α-emulsan inthe emulsification of hydrocarbons is dependent on the relativeconcentrations of aliphatic and aromatic (or cyclic) compounds in thehydrocarbon substrate. For example, the ability of emulsan to emulsifykerosene and gasoline was enhanced greatly by 2-methylnaphthalene butnot by hexadecane. The requirement that the hydrocarbon substratecontain both aliphatic and aromatic (or cyclic) components was furthersupported by the results obtained in the emulsification of mixtures ofcolumn fractionated crude oil. Although crude oil itself is emulsifiedby emulsan, none of the fractions were good substrates by themselves.Mixtures containing one fraction rich in aliphatics (Fraction 1) and theother rich in aromatics (Fractions 2 or 3) were efficiently emulsified.

13.16. CLEANING OIL-CONTAMINATED VESSELS

Aqueous solutions in sea water or fresh water (the latter containing asuitable divalent cation, such as magnesium) of α-emulsans are excellentemulsifying agents for cleaning and recovering hydrocarbonaceousresidues, including residual crude oil, from oil-contaminated tankers,barges, storage tanks, tank cars and trucks, pipelines and othercontainers used to transport or store crude oil or petroleum fractions.Washing the oil-contaminated surfaces of such vessels with an aqueoussolution containing from about 10 mcg/ml to about 20 mg/ml of α-emulsanreadily forms an oil-in-water emulsion of such hydrocarbonaceousresidues provided that the solution contains from about 1 to about 100mM, and preferably from about 5 to about 40 mM, of at least one suitabledivalent cation, which are normally present in sea water and "hard" tapwater. Moreover, the α-emulsan need not be purified, since a cell-freefermentation broth containing α-emulsans resulting from growingAcinetobacter Sp. ATCC 31012 on a suitable medium can be used directlyor after suitable dilution.

Using the data which is set forth above in Sections 8 and 9, processescan be designed to clean any oil-contaminated vessel and to recover thehydrocarbonaceous residue from the resultant oil-in-water emulsion,either by breaking the emulsion physically or chemically. Depending uponthe amount and composition of the oil or hydrocarbonaceous residue to becleaned, the aggregate amount of α-emulsan may be as low as 1 part byweight (dry weight basis) per 1,000 to 10,000 parts by weight ofhydrocarbon, the higher concentrations of α-emulsan yielding more stableemulsions.

To show the use of the cell-free fermentation broth as an emulsifyingagent for such cleaning, Acinetobacter Sp. ATCC 31012 was cultivated ina 15 liter glass fermenter containing 122 g of dibasic potassiumphosphate [K₂ HPO₄.3H₂ O], 40 g of monobasic potassium phosphate, 1.33 gof magnesium sulfate [MgSO₄.7H₃ O], 13.3 g of urea and deionized waterto a final volume of 10 liters. The medium was sterilized for 30 minutesat 121° C., after which 200 ml of absolute ethanol (2% by volume) wasadded. The final pH of the ethanol-salts medium was 7.0. After themedium had cooled to 30° C., 500 ml of a late exponential culture ofAcinetobacter Sp. ATCC 31012 grown in the same medium was added to theglass fermenter and the culture maintained at 30° C., with an air flowof 3.5 liters per minute and an agitation speed of 200 rpm (no baffles).During the course of fermentation the pH dropped to 6.0. Throughout thefermentation, foam was controlled by periodic addition of siliconedefoamer (in the form of a spray).

Under these conditions, the fermentation broth contained 260 units perml of α-emulsan after 72 hours and 7.4 g liter of biomass (dried at 90°C. for 16 hours). After removal of the cells by centrifugation orfiltration, the resultant cell-free fermentation broth could be used towash crude oil from the oil-contaminated surface of a steel containerwhich simulated the inner wall of a tank which had been emptied of crudeoil.

13.17. EFFECT OF MOBILITY CONTROL POLYSACCHARIDES ON EMULSION FORMATIONWITH EMULSANS

The bacterial exocellular heteropolysaccharide (XANFLD SFL 14630)produced by the Kelco Division of Merck & Co., Inc., which has beenrecommended as a mobility control polymer for enhanced oil recovery, wastested in varying concentrations in conjunction with 20 mcg/ml ofα-emulsan to determine the effect of such material on the emulsificationof gas-oil. In each of these tests, 0.1 ml of Gach-Saran gas-oil wasadded to 125 ml Ehrlenmeyer flasks containing 7.5 ml of Tris-Mg buffer[50 mM tris(hydroxymethyl)aminomethane hydrochloride, pH 7.2; 10 mMmagnesium chloride], 20 mcg/ml of α-emulsan and varying concentrationsof the mobility control polysaccharide. Several tests were also runwithout the α-emulsan to determine whether the mobility control polymeremulsified the hydrocarbon.

The flasks were agitated by gyratory shaking (280 rpm) in a NewBrunswick G24 incubator shaker for one hour at 30° C. Contents of theflasks were then transferred to Klett tubes for measurement of turbidityin a Klett-Summerson colorimeter fitted with a green filter. Readingswere taken after standing undisturbed for 10 minutes. The results ofthese tests, which are summarized in Table XV, are expressed as thepercentage increase (+) or decrease (-) in the turbidity of the emulsionresulting from the addition of varying concentrations of the mobilitycontrol polymer.

                  TABLE XV                                                        ______________________________________                                        Effect of Mobility Control Polysaccharide                                     on Emulsion Formation                                                         Mobility Control                                                                             α-Emulsan                                                                          Relative Emulsifying                                Polysaccharide (mcg/ml)                                                                      (mcg/ml)   Ability (%)                                         ______________________________________                                         1             20         -17.0                                                2             20         +3.1                                                 5             20         -7.3                                                10             20         +41.7                                               20             20         +27.2                                               40             20         +20.6                                               10-150         None       No Activity                                         ______________________________________                                    

As shown in Table XV, it appears that the use of the mobility controlpolysaccharide in conjunction with the emulsan is capable of stimulatingemulsifying activity by about 40% at a concentration of 10 mcg/ml, whichsuggests the potential advantages for using both additives in achemically-augmented "slug" to be injected into a petroleum reservoirfor enhanced oil recovery. By itself, however, this mobility controlpolymer had no ability to emulsify the hydrocarbon.

13.18. ADSORPTION OF EMULSANS ON CLAYS

Because of the importance of aluminosilicate clays, such as kaolin andbentonite, in many industrial and petroleum production and refiningprocesses, a series of tests was conducted to determine whether emulsansadsorped onto the surface of such aluminosilicate clays. Bentonite wasselected for these tests, since it contains up to 90% by weight ofmontmorillonite, the structure of which corresponds to the theoreticalformula (OH)₄ Si₈ Al₄ O₂₀.xH₂ O and is responsible for its high sorptivepower and ion-exchange capacity.

The theoretical treatment of adsorption from a mixed solution issomewhat complicated, since it involves competition between solutes andsolvents for the solid surface. In these tests, adsorption from solutionwas analyzed by the Freundlich equation: ##EQU1## where x represents theamount of solute adsorped by the mass m of solid, C represents thesolute concentration and a and n are experimentally-determinedconstants. Experimentally, x=(C_(o) --C)V, where C_(o) and C are theinitial and equilibrium solute concentrations, respectively, and V isthe volume of solution in contact with the sorbent. In this case, anapparent adsorption isotherm can be expressed if x/m is plotted againstequilibrium solute concentration.

In each of these tests, the emulsan used was an α-emulsan purified inaccordance with the ammonium sulfate fractionation technique describedabove in Section 13.10. Prior to drying, the α-emulsan contained about7% by weight of protein, about 16% by weight of ash and about 38% byweight of moisture. Aqueous solutions of this α-emulsan were prepared bydissolving the dry emulsan in 0.02 M solutions of Tris-Mg buffer [20 mMtris-(hydroxymethyl)aminomethane HCl containing 10 mM magnesiumsulfate]. Nonactivated, technical grade bentonite was used as thesorbent.

Adsorption of α-emulsan from a given volume of solution on a given massof bentonite was carried out in 100 ml or 50 ml Ehrlenmeyer flasks, withshaking for 1 hour at 100 strokes per minute. The equilibrium solutionswere separated from the bentonite by centrifugation or filtration.Emulsan assays were performed by the standard assay technique. Theresults of the tests are summarized in Table XVI.

                  TABLE XVI                                                       ______________________________________                                        Adsorption of α-Emulsan on Bentonite                                    Bentonite                                                                              α-Emulsan                                                                             (mg/ml)                                                (mg)     C.sub.o       C        % Bound                                       ______________________________________                                        10       0.11          0.032    71                                            20       0.11          0.028    75                                            25       0.10          0.006    94                                            40       0.11          0.004    96                                            60       0.11          <0.001   >99                                           ______________________________________                                    

The data contained in Table XVI show that the adsorption of α-emulsan tobentonite is a function of bentonite concentration. About 70% of theemulsan is adsorbed when the ratio of bentonite to emulsan is 100:1,while more than 95% of the emulsan is adsorbed at ratios of 400:1 orhigher.

13.19. FLOCCULATION OF CLAYS BY EMULSANS

Adsorption of emulsan on bentonite results in flocculation of suspendedparticles of the clay, with sedimentation occurring about 5 to 10 timesfaster than in the absence of emulsan. To a solution of 50 ml sea waterand 50 ml Tris buffer containing 100 mcg/ml of α-emulsan was added 1.6 gof non-activated, technical-grade bentonite with mixing, and theresultant dispersion was then poured into a calibrated glass cylinderand allowed to settle at room temperature. As a control, a parallelexperiment was conducted without using the α-emulsan.

The results of these tests, which are graphically illustrated in FIG.18, show that the dilute (100 ppm) solution of α-emulsan enhanced therate of sedimentation by a factor of five over that obtained in thecontrol. More importantly, the supernatant fluid obtained following theemulsan-mediated flocculation was clear, while the supernatant fluidobtained in the control remained opalescent even after prolongedstanding.

13.20. FLOCCULATION OF CLAYS BY PROEMULSANS

Proemulsans are even more effective than emulsans in the flocculation ofsuspended particles of bentonite. Table XVII summarizes an experiment inwhich the flocculating of 0.4 g bentonite in 14 ml of either Trisbuffer, pH 7.26, or phosphate buffer, pH 6.5, was measured in thepresence of α-emulsan, proemulsan and no addition. The finalconcentration of α-emulsan was 0.05 mg per ml, whereas the finalconcentration of proemulsan was 0.045 mg/ml. After vigorous shaking for2 minutes, the suspension was centrifuged at 25,000 rpm for 60 minutes.The data presented in Table XVIII were obtained by measuring theclarified upper layer in the centrifuge tube. Similar results wereobtained with ψ-emulsan as with proemulsan.

                  TABLE XVII                                                      ______________________________________                                        Flocculation of Bentonite by α-Emulsan and Proemulsan                           Volume of Clear                                                               Upper Layer (ml)                                                      Sample        PH 6.5   pH 7.26                                                ______________________________________                                        1. No addition                                                                              1.1.sup.a                                                                              1.1                                                    2. α-Emulsan                                                                          1.8.sup.a                                                                              2.0                                                    3. Proemulsan 3.0      4.6                                                    ______________________________________                                         .sup.a The upper layer was opalescent.                                   

13.21. BREAKING EMULSAN-INDUCED EMULSIONS

Since emulsans form stable oil-in-water emulsions and, moreover, sinceemulsans are adsorbed onto bentonite, a series of tests were conductedto determine the behavior of such emulsan-induced emulsions in thepresence of bentonite. In one test, an emulsion of Agha Jari crude oil(1 ml in 10 ml sea water) containing about 0.1 mg/ml of α-emulsan wasprepared by the standard technique of adding the oil to the solution ofα-emulsan in a flask and agitating the flask by gyratory shaking for onehour at room temperature. After 2 days, 1 g of preswelled bentonite wasadded to the stable emulsion and the dispersion was shaken intensivelyfor about 20 seconds, after which it was transferred to a tube andallowed to settle. After 15 minutes, a breakage of the emulsion wasobserved. After 20 hours, two layers had separated, the upper layerbeing clear while the lower layer was a gel-like sediment which occupiedabout one-half the prior volume of the emulsion

In another test, an emulsion of Agha Jari crude oil (0.1 ml) in 7.5 mlTris-Mg buffer solution [50 mM tris-(hydroxymethyl)aminomethanehydrochloride, pH 7.2; 10 mM magnesium chloride] containing 0.08 mg/mlof α-emulsan was prepared by the standard technique as before. As acontrol, 0.1 ml of the crude oil in 7.5 ml of buffer solution was shakenunder the same conditions. Both samples were transferred to tubescontaining 0.5 g of bentonite, shaken for 30 seconds and the contentsallowed to settle. After 15 hours, there was a complete breakage of theemulsan-induced emulsion. Moreover, the flocculated sediment formed inthe presence of α-emulsan was two times larger by volume than thesediment from the control test.

13.22. REMOVAL OF OIL FROM SAND BY EMULSAN

One gram of white sand was preadsorbed with either 0.1 ml, 0.2 ml, or0.3 ml (saturated) Darius crude oil (light weight Persian crude) induplicate. The sand samples were then transferred to 100 ml Ehrlenmeyerflasks containing 10 ml Tris-Mg buffer [50 mMtris-(hydroxymethyl)aminomethane hydrochloride, pH 7.2; 10 mM magnesiumsulfate]. To one each of the samples containing 0.1 ml, 0.2 ml, or 0.3ml of the crude oil was added α-emulsan at a final concentration of 0.1mg/ml. The remaining three samples (without the emulsan) served ascontrols. The samples were shaken at 140 strokes per minute for 30minutes at 30° C. in a shaking water bath.

Following the shaking, the samples were allowed to settle for one hour,and the aqueous phase was separated from the sand by decantation. Eachof the sand samples was washed twice with 10 ml Tris-Mg buffer, and thedecanted washed fluids combined. The sand samples and the aqueous phase(together with the wash fluids) were each separately extracted withdiethyl ether and the ether extract dried under nitrogen in taredflasks. Table XVIII summarizes the results of these tests, where theamount of oils removed by α-emulsan is measured as the amount ofether-extracted material in the water phase and the amount of oilremaining on the sand is measured as the amount of ether-solublematerial extracted from the washed sand.

                  TABLE XVIII                                                     ______________________________________                                        Effect of α-Emulsan on                                                  Removal of Crude Oil from Sand                                                                   Oil       Oil                                                                 Removed   Remaining                                        Crude Oil                                                                              Emulsan   from      on       Removal                                 on Sand (ml)                                                                           (mg/ml)   Sand (mg) Sand (mg)                                                                              (%)                                     ______________________________________                                        0.1      --        <5         65      <10                                     0.1      0.1       57        <5       >90                                     0.2      --        15        108      12                                      0.2      0.1       96         12      89                                      0.3      --        33        172      16                                      0.3      0.1       165        14      92                                      ______________________________________                                    

The effect of α-emulsan in removing oil from sand is clearlydemonstrated in Table XVIII. In the presence of 0.1 mg/ml, over 90% ofthe crude oil was removed. This is probably a lower estimate since etherextraction of sand particles which had not settled before the initialseparation of the phases would contribute to the overall amount ofmaterial extracted from the aqueous phase in the control. Very little(<10%) of the oil was removed without shaking. During these tests, itwas observed that the solubilized oil emulsified in those samples towhich α-emulsan was added. Moreover, the addition of the α-emulsan to aflask containing sand and buffer prior to preadsorption of the oilprevented the subsequent adsorption of oil to the sand during theshaking.

From the data contained in Table XVIII, it is clear that emulsans may beused in enhanced recovery processes for recovering oil which iscontained in sand or sandstone formations, in which processes achemically-augmented "slug" comprising water or brine and one or moreadded chemicals is injected into a petroleum reservoir located in a sandor sandstone formation and is displaced through the reservoir to recovercrude oil. In addition, dilute solutions of emulsans (which arebiodegradable) may be used in oil spill management to emulsify oilspills deposited on beach sand so that the oil may be dispersed andsubsequently microbiologically degraded.

13.23. REMOVAL OF OIL FROM LIMESTONE BY EMULSAN

A series of tests was conducted to determine the ability of emulsan toremove oil from limestone, since enhanced oil recovery processes basedon chemical flooding of petroleum reservoirs located in reservoirformations (in which processes a chemically-augmented "slug" of water orbrine and one or more added chemicals is injected into a petroleumreservoir in a limestone formation and is displaced through thereservoir to recover crude oil) will require efficient emulsifierscapable of removing oil from limestone, which chemically is calciumcarbonate.

Four 4-gram samples of calcium carbonate (crushed limestone) were eachpreadsorbed with 0.8 g Aghi Jari crude oil. The oil-impregnatedlimestone samples were then transferred to 100 ml Ehrlenmeyer flaskscontaining 20 ml Tris-Mg buffer [50 mM tris-(hydroxymethyl)aminomethanehydrochloride, pH 7.2; 10 mM magnesium sulfate]. To each of three of thesamples was added varying amounts of α-emulsan (2, 5 and 10 mg,respectively) while the remaining sample (without the emulsan) served asa control. The samples were shaken at 140 strokes per minute for 30minutes at 30° C. in a shaking water bath.

Following the shaking, the samples were allowed to settle for 1 hour,and the aqueous phase was separated from the limestone by decantation.Each of the limestone samples was washed twice with 10 ml Tris-Mgbuffer, and the decanted washed fluid combined. The limestone samplesand the aqueous phase (together with the wash fluids) were eachseparately extracted with diethyl ether and the ether extract driedunder nitrogen in tared flasks. Table XIX summarizes the results ofthese tests, where the amount of oils removed by α-emulsan is measuredas the amount of ether-extracted material in the water phase and theamount of oil remaining on the limestone was calculated by difference.

                  TABLE XIX                                                       ______________________________________                                        Effect of α-Emulsan on                                                  Removal of Crude Oil from Limestone                                                 Emulsan   Oil Removed Oil Remaining                                                                           Removal                                 Sample                                                                              (mg/ml)   (g)         (g)       (%)                                     ______________________________________                                        A     --        0.06        0.74      14                                      B     0.1       0.71        0.09      89                                      C     0.25      0.74        0.06      93                                      D     0.5       0.78        0.02      98                                      ______________________________________                                    

The effect of α-emulsan in removing oil from limestone is clearlydemonstrated in Table XIX. In the presence of 0.1 mg/ml, over 89% of thecrude oil was removed; at α-emulsan concentrations of 0.5 mg/ml, over98% of the crude oil was removed. As in the case of the tests describedabove in Section 13.22, this is probably a lower estimate since etherextraction of limestone particles which had not settled before theinitial separation of the phases would contribute to the overall amountof material extracted from the aqueous phase in the control.

Because emulsans and particularly the α-emulsans (on a weight-for-weightbasis) are probably the most efficient oil-in-water emulsifiers inexistence and because these extracellular lipopolysaccharides toleraterelatively high concentrations of sodium chloride without losing theiremulsification activity, it is expected that emulsans will be widelyused in all enhanced oil recovery techniques for freeing oil fromlimestone formations.

We claim:
 1. Polyanionic heteropolysaccharide biopolymers in which (a)substantially all of the sugar moieties are N-acylated aminosugars, aportion of which is N-acylated-D-galactosamine and another portion ofwhich is N-acylated aminouronic acid, a part of the N-acyl groups ofsuch heteropolysaccharide being N-3-hydroxydodecanoyl groups; and (b) atleast 0.2 micromoles per milligram of such heteropolysaccharideconsisting of fatty acid esters in which (1) the fatty acids containabout 10 to about 18 carbon atoms and (2) about 50 percent by weight orhigher of such fatty acids are composed of 2-hydroxydodecanoic acid and3-hydroxydodecanoic acid.
 2. Polyanionic heteropolysaccharidebiopolymers according to claim 1, in which the fatty acids of the0-lipoacyl esters have an average equivalent weight from about 200 toabout
 230. 3. Polyanionic heteropolysaccharide biopolymers according toclaim 1, in which the fatty acids esters constitute at least 0.5micromoles per milligram of such heteropolysaccharide.
 4. Polyanionicheteropolysaccharide biopolymers according to claim 1, in which thefatty acid esters constitute from about 0.5 to about 0.75 micromoles permilligram of such heteropolysaccharide.
 5. Polyanionicheteropolysaccharide biopolymers according to claim 1, in which theheteropolysaccharide is in the form of a salt of a divalent cation. 6.Polyanionic heteropolysaccharide biopolymers according to any of claims1, 2, 3, 4, or 5 in which the ratio of 2-hydroxydodecanoic acid to3-hydroxydodecanoic acid in the 0-lipoacyl portion of suchheteropolysaccharide is in the range from about 1:4 to about 1:1. 7.Polyanionic heteropolysaccharide biopolymers according to any of claims1, 2, 3, 4, or 5 in which the ratio of 2-hydroxydodecanoic acid to3-hydroxydodecanoic acid in the 0-lipoacyl of such heteropolysaccharideis in the range from about 1:4 to about 1:2.
 8. Polyanionicheteropolysaccharide biopolymers according to any of claims 1, 2, 3, 4,or 5, in which the heteropolysaccharide is in the form of its magnesiumsalt.
 9. Polyanionic heteropolysaccharide biopolymers according to anyof claims 1, 2, 3, 4, or 5, in which the heteropolysaccharide biopolymeris complexed with a protein and is characterized by a SpecificEmulsification Activity of about 200 units per milligram or higher,where one unit per milligram of Specific Emulsification Activity isdefined as that amount of emulsifying activity per milligram of theprotein-complexed biopolymer which yields 100 Klett absorption unitsusing a standard hydrocarbon mixture consisting of 0.1 ml of 1:1 (v/v)hexadecane/2-methylnaphthalene and 7.5 ml of Tris-Mg buffer. 10.Polyanionic heteropolysaccharide biopolymers of claim 1 furthercomprising divalent metal, ammonium and quaternary ammonium salts ofsuch polyanionic heteropolysaccharide biopolymers.